Intact yeast cells treated with alkali cations took up plasmid DNA. Li+, Cs+, Rb+, K+, and Na+ were effective in inducing competence. Conditions for the transformation of Saccharomyces cerevisiae D<em>1</em>3-<em>1</em>A with plasmid YRp7 were studied in detail with CsCl. The optimum incubation time was <em>1</em> h, and the optimum cell concentration was 5 x <em>1</em>0(7) cells per <em>ml</em>. The optimum concentration of Cs+ was <em>1</em>.0 M. Transformation efficiency increased with increasing concentrations of plasmid DNA. Polyethylene glycol was absolutely required. Heat pulse and various polyamines or basic proteins stimulated the uptake of plasmid DNA. Besides circular DNA, linear plasmid DNA was also taken up by Cs+-treated yeast cells, although the uptake efficiency was considerably reduced. The transformation efficiency with Cs+ or Li+ was comparable with that of conventional protoplast methods for a plasmid containing ars<em>1</em>, although not for plasmids containing a 2 microns origin replication.

Traditional approaches to mechanical ventilation use tidal volumes of 10 to 15 ml per kilogram of body weight and may cause stretch-induced lung injury in patients with acute lung injury and the acute respiratory distress syndrome. We therefore conducted a trial to determine whether ventilation with lower tidal volumes would improve the clinical outcomes in these patients.

Patients with acute lung injury and the acute respiratory distress syndrome were enrolled in a multicenter, randomized trial. The trial compared traditional ventilation treatment, which involved an initial tidal volume of 12 ml per kilogram of predicted body weight and an airway pressure measured after a 0.5-second pause at the end of inspiration (plateau pressure) of 50 cm of water or less, with ventilation with a lower tidal volume, which involved an initial tidal volume of 6 ml per kilogram of predicted body weight and a plateau pressure of 30 cm of water or less. The primary outcomes were death before a patient was discharged home and was breathing without assistance and the number of days without ventilator use from day 1 to day 28.

The trial was stopped after the enrollment of 861 patients because mortality was lower in the group treated with lower tidal volumes than in the group treated with traditional tidal volumes (31.0 percent vs. 39.8 percent, P=0.007), and the number of days without ventilator use during the first 28 days after randomization was greater in this group (mean [+/-SD], 12+/-11 vs. 10+/-11; P=0.007). The mean tidal volumes on days 1 to 3 were 6.2+/-0.8 and 11.8+/-0.8 ml per kilogram of predicted body weight (P<0.001), respectively, and the mean plateau pressures were 25+/-6 and 33+/-8 cm of water (P<0.001), respectively.

In patients with acute lung injury and the acute respiratory distress syndrome, mechanical ventilation with a lower tidal volume than is traditionally used results in decreased mortality and increases the number of days without ventilator use.

Control elements of the tetracycline-resistance operon encoded in Tn<em>1</em>0 of Escherichia coli have been utilized to establish a highly efficient regulatory system in mammalian cells. By fusing the tet repressor with the activating domain of virion protein <em>1</em>6 of herpes simplex virus, a tetracycline-controlled transactivator (tTA) was generated that is constitutively expressed in HeLa cells. This transactivator stimulates transcription from a minimal promoter sequence derived from the human cytomegalovirus promoter IE combined with tet operator sequences. Upon integration of a luciferase gene controlled by a tTA-dependent promoter into a tTA-producing HeLa cell line, high levels of luciferase expression were monitored. These activities are sensitive to tetracycline. Depending on the concentration of the antibiotic in the culture medium (0-<em>1</em> microgram/<em>ml</em>), the luciferase activity can be regulated over up to five orders of magnitude. Thus, the system not only allows differential control of the activity of an individual gene in mammalian cells but also is suitable for creation of "on/off" situations for such genes in a reversible way.

OBJECTIVE

To provide an update to the "Surviving Sepsis Campaign Guidelines for Management of Severe Sepsis and Septic Shock," last published in 2008.

METHODS

A consensus committee of 68 international experts representing 30 international organizations was convened. Nominal groups were assembled at key international meetings (for those committee members attending the conference). A formal conflict of interest policy was developed at the onset of the process and enforced throughout. The entire guidelines process was conducted independent of any industry funding. A stand-alone meeting was held for all subgroup heads, co- and vice-chairs, and selected individuals. Teleconferences and electronic-based discussion among subgroups and among the entire committee served as an integral part of the development.

METHODS

The authors were advised to follow the principles of the Grading of Recommendations Assessment, Development and Evaluation (GRADE) system to guide assessment of quality of evidence from high (A) to very low (D) and to determine the strength of recommendations as strong (<em>1</em>) or weak (2). The potential drawbacks of making strong recommendations in the presence of low-quality evidence were emphasized. Some recommendations were ungraded (UG). Recommendations were classified into three groups: <em>1</em>) those directly targeting severe sepsis; 2) those targeting general care of the critically ill patient and considered high priority in severe sepsis; and 3) pediatric considerations.

RESULTS

Key recommendations and suggestions, listed by category, include: early quantitative resuscitation of the septic patient during the first 6 hrs after recognition (<em>1</em>C); blood cultures before antibiotic therapy (<em>1</em>C); imaging studies performed promptly to confirm a potential source of infection (UG); administration of broad-spectrum antimicrobials therapy within <em>1</em> hr of recognition of septic shock (<em>1</em>B) and severe sepsis without septic shock (<em>1</em>C) as the goal of therapy; reassessment of antimicrobial therapy daily for de-escalation, when appropriate (<em>1</em>B); infection source control with attention to the balance of risks and benefits of the chosen method within <em>1</em>2 hrs of diagnosis (<em>1</em>C); initial fluid resuscitation with crystalloid (<em>1</em>B) and consideration of the addition of albumin in patients who continue to require substantial amounts of crystalloid to maintain adequate mean arterial pressure (2C) and the avoidance of hetastarch formulations (<em>1</em>C); initial fluid challenge in patients with sepsis-induced tissue hypoperfusion and suspicion of hypovolemia to achieve a minimum of 30 mL/kg of crystalloids (more rapid administration and greater amounts of fluid may be needed in some patients) (<em>1</em>C); fluid challenge technique continued as long as hemodynamic improvement, as based on either dynamic or static variables (UG); norepinephrine as the first-choice vasopressor to maintain mean arterial pressure ≥ 65 mm Hg (<em>1</em>B); epinephrine when an additional agent is needed to maintain adequate blood pressure (2B); vasopressin (0.03 U/min) can be added to norepinephrine to either raise mean arterial pressure to target or to decrease norepinephrine dose but should not be used as the initial vasopressor (UG); dopamine is not recommended except in highly selected circumstances (2C); dobutamine infusion administered or added to vasopressor in the presence of a) myocardial dysfunction as suggested by elevated cardiac filling pressures and low cardiac output, or b) ongoing signs of hypoperfusion despite achieving adequate intravascular volume and adequate mean arterial pressure (<em>1</em>C); avoiding use of intravenous hydrocortisone in adult septic shock patients if adequate fluid resuscitation and vasopressor therapy are able to restore hemodynamic stability (2C); hemoglobin target of 7-9 g/dL in the absence of tissue hypoperfusion, ischemic coronary artery disease, or acute hemorrhage (<em>1</em>B); low tidal volume (<em>1</em>A) and limitation of inspiratory plateau pressure (<em>1</em>B) for acute respiratory distress syndrome (ARDS); application of at least a minimal amount of positive end-expiratory pressure (PEEP) in ARDS (<em>1</em>B); higher rather than lower level of PEEP for patients with sepsis-induced moderate or severe ARDS (2C); recruitment maneuvers in sepsis patients with severe refractory hypoxemia due to ARDS (2C); prone positioning in sepsis-induced ARDS patients with a PaO2/FIO2 ratio of ≤ <em>1</em>00 mm Hg in facilities that have experience with such practices (2C); head-of-bed elevation in mechanically ventilated patients unless contraindicated (<em>1</em>B); a conservative fluid strategy for patients with established ARDS who do not have evidence of tissue hypoperfusion (<em>1</em>C); protocols for weaning and sedation (<em>1</em>A); minimizing use of either intermittent bolus sedation or continuous infusion sedation targeting specific titration endpoints (<em>1</em>B); avoidance of neuromuscular blockers if possible in the septic patient without ARDS (<em>1</em>C); a short course of neuromuscular blocker (no longer than 48 hrs) for patients with early ARDS and a Pao2/Fio2 < <em>1</em>50 mm Hg (2C); a protocolized approach to blood glucose management commencing insulin dosing when two consecutive blood glucose levels are>> <em>1</em>80 mg/dL, targeting an upper blood glucose ≤ <em>1</em>80 mg/dL (<em>1</em>A); equivalency of continuous veno-venous hemofiltration or intermittent hemodialysis (2B); prophylaxis for deep vein thrombosis (<em>1</em>B); use of stress ulcer prophylaxis to prevent upper gastrointestinal bleeding in patients with bleeding risk factors (<em>1</em>B); oral or enteral (if necessary) feedings, as tolerated, rather than either complete fasting or provision of only intravenous glucose within the first 48 hrs after a diagnosis of severe sepsis/septic shock (2C); and addressing goals of care, including treatment plans and end-of-life planning (as appropriate) (<em>1</em>B), as early as feasible, but within 72 hrs of intensive care unit admission (2C). Recommendations specific to pediatric severe sepsis include: therapy with face mask oxygen, high flow nasal cannula oxygen, or nasopharyngeal continuous PEEP in the presence of respiratory distress and hypoxemia (2C), use of physical examination therapeutic endpoints such as capillary refill (2C); for septic shock associated with hypovolemia, the use of crystalloids or albumin to deliver a bolus of 20 mL/kg of crystalloids (or albumin equivalent) over 5 to <em>1</em>0 mins (2C); more common use of inotropes and vasodilators for low cardiac output septic shock associated with elevated systemic vascular resistance (2C); and use of hydrocortisone only in children with suspected or proven "absolute"' adrenal insufficiency (2C).

CONCLUSIONS

Strong agreement existed among a large cohort of international experts regarding many level <em>1</em> recommendations for the best care of patients with severe sepsis. Although a significant number of aspects of care have relatively weak support, evidence-based recommendations regarding the acute management of sepsis and septic shock are the foundation of improved outcomes for this important group of critically ill patients.

BACKGROUND

Estimation of glomerular filtration rate (GFR) is limited by differences in creatinine generation among ethnicities. Our previously reported GFR-estimating equations for Japanese had limitations because all participants had a GFR less than 90 mL/min/1.73 m2 and serum creatinine was assayed in different laboratories.

METHODS

Diagnostic test study using a prospective cross-sectional design. New equations were developed in 413 participants and validated in 350 participants. All samples were assayed in a central laboratory.

METHODS

Hospitalized Japanese patients in 80 medical centers. Patients had not participated in the previous study.

METHODS

Measured GFR (mGFR) computed from inulin clearance.

METHODS

Estimated GFR (eGFR) by using the modified isotope dilution mass spectrometry (IDMS)-traceable 4-variable Modification of Diet in Renal Disease (MDRD) Study equation using the previous Japanese Society of Nephrology Chronic Kidney Disease Initiative (JSN-CKDI) coefficient of 0.741 (equation 1), the previous JSN-CKDI equation (equation 2), and new equations derived in the development data set: modified MDRD Study using a new Japanese coefficient (equation 3), and a 3-variable Japanese equation (equation 4).

METHODS

Performance of equations was assessed by means of bias (eGFR - mGFR), accuracy (percentage of estimates within 15% or 30% of mGFR), root mean squared error, and correlation coefficient.

RESULTS

In the development data set, the new Japanese coefficient was 0.808 (95% confidence interval, 0.728 to 0.829) for the IDMS-MDRD Study equation (equation 3), and the 3-variable Japanese equation (equation 4) was eGFR (mL/min/1.73 m2) = 194 x Serum creatinine(-1.094) x Age(-0.287) x 0.739 (if female). In the validation data set, bias was -1.3 +/- 19.4 versus -5.9 +/- 19.0 mL/min/1.73 m2 (P = 0.002), and accuracy within 30% of mGFR was 73% versus 72% (P = 0.6) for equation 3 versus equation 1 and -2.1 +/- 19.0 versus -7.9 +/- 18.7 mL/min/1.73 m(2) (P < 0.001) and 75% versus 73% (P = 0.06) for equation 4 versus equation 2 (P = 0.06), respectively.

CONCLUSIONS

Most study participants had chronic kidney disease, and some may have had changing GFRs.

CONCLUSIONS

The new Japanese coefficient for the modified IDMS-MDRD Study equation and the new Japanese equation are more accurate for the Japanese population than the previously reported equations.

OBJECTIVE

To provide an update to the "Surviving Sepsis Campaign Guidelines for Management of Severe Sepsis and Septic Shock," last published in 2008.

METHODS

A consensus committee of 68 international experts representing 30 international organizations was convened. Nominal groups were assembled at key international meetings (for those committee members attending the conference). A formal conflict of interest policy was developed at the onset of the process and enforced throughout. The entire guidelines process was conducted independent of any industry funding. A stand-alone meeting was held for all subgroup heads, co- and vice-chairs, and selected individuals. Teleconferences and electronic-based discussion among subgroups and among the entire committee served as an integral part of the development.

METHODS

The authors were advised to follow the principles of the Grading of Recommendations Assessment, Development and Evaluation (GRADE) system to guide assessment of quality of evidence from high (A) to very low (D) and to determine the strength of recommendations as strong (<em>1</em>) or weak (2). The potential drawbacks of making strong recommendations in the presence of low-quality evidence were emphasized. Recommendations were classified into three groups: (<em>1</em>) those directly targeting severe sepsis; (2) those targeting general care of the critically ill patient and considered high priority in severe sepsis; and (3) pediatric considerations.

RESULTS

Key recommendations and suggestions, listed by category, include: early quantitative resuscitation of the septic patient during the first 6 h after recognition (<em>1</em>C); blood cultures before antibiotic therapy (<em>1</em>C); imaging studies performed promptly to confirm a potential source of infection (UG); administration of broad-spectrum antimicrobials therapy within <em>1</em> h of the recognition of septic shock (<em>1</em>B) and severe sepsis without septic shock (<em>1</em>C) as the goal of therapy; reassessment of antimicrobial therapy daily for de-escalation, when appropriate (<em>1</em>B); infection source control with attention to the balance of risks and benefits of the chosen method within <em>1</em>2 h of diagnosis (<em>1</em>C); initial fluid resuscitation with crystalloid (<em>1</em>B) and consideration of the addition of albumin in patients who continue to require substantial amounts of crystalloid to maintain adequate mean arterial pressure (2C) and the avoidance of hetastarch formulations (<em>1</em>B); initial fluid challenge in patients with sepsis-induced tissue hypoperfusion and suspicion of hypovolemia to achieve a minimum of 30 mL/kg of crystalloids (more rapid administration and greater amounts of fluid may be needed in some patients (<em>1</em>C); fluid challenge technique continued as long as hemodynamic improvement is based on either dynamic or static variables (UG); norepinephrine as the first-choice vasopressor to maintain mean arterial pressure ≥65 mmHg (<em>1</em>B); epinephrine when an additional agent is needed to maintain adequate blood pressure (2B); vasopressin (0.03 U/min) can be added to norepinephrine to either raise mean arterial pressure to target or to decrease norepinephrine dose but should not be used as the initial vasopressor (UG); dopamine is not recommended except in highly selected circumstances (2C); dobutamine infusion administered or added to vasopressor in the presence of (a) myocardial dysfunction as suggested by elevated cardiac filling pressures and low cardiac output, or (b) ongoing signs of hypoperfusion despite achieving adequate intravascular volume and adequate mean arterial pressure (<em>1</em>C); avoiding use of intravenous hydrocortisone in adult septic shock patients if adequate fluid resuscitation and vasopressor therapy are able to restore hemodynamic stability (2C); hemoglobin target of 7-9 g/dL in the absence of tissue hypoperfusion, ischemic coronary artery disease, or acute hemorrhage (<em>1</em>B); low tidal volume (<em>1</em>A) and limitation of inspiratory plateau pressure (<em>1</em>B) for acute respiratory distress syndrome (ARDS); application of at least a minimal amount of positive end-expiratory pressure (PEEP) in ARDS (<em>1</em>B); higher rather than lower level of PEEP for patients with sepsis-induced moderate or severe ARDS (2C); recruitment maneuvers in sepsis patients with severe refractory hypoxemia due to ARDS (2C); prone positioning in sepsis-induced ARDS patients with a PaO (2)/FiO (2) ratio of ≤<em>1</em>00 mm Hg in facilities that have experience with such practices (2C); head-of-bed elevation in mechanically ventilated patients unless contraindicated (<em>1</em>B); a conservative fluid strategy for patients with established ARDS who do not have evidence of tissue hypoperfusion (<em>1</em>C); protocols for weaning and sedation (<em>1</em>A); minimizing use of either intermittent bolus sedation or continuous infusion sedation targeting specific titration endpoints (<em>1</em>B); avoidance of neuromuscular blockers if possible in the septic patient without ARDS (<em>1</em>C); a short course of neuromuscular blocker (no longer than 48 h) for patients with early ARDS and a PaO (2)/FI O (2) (<em>1</em>50 mm Hg (2C); a protocolized approach to blood glucose management commencing insulin dosing when two consecutive blood glucose levels are>><em>1</em>80 mg/dL, targeting an upper blood glucose ≤<em>1</em>80 mg/dL (<em>1</em>A); equivalency of continuous veno-venous hemofiltration or intermittent hemodialysis (2B); prophylaxis for deep vein thrombosis (<em>1</em>B); use of stress ulcer prophylaxis to prevent upper gastrointestinal bleeding in patients with bleeding risk factors (<em>1</em>B); oral or enteral (if necessary) feedings, as tolerated, rather than either complete fasting or provision of only intravenous glucose within the first 48 h after a diagnosis of severe sepsis/septic shock (2C); and addressing goals of care, including treatment plans and end-of-life planning (as appropriate) (<em>1</em>B), as early as feasible, but within 72 h of intensive care unit admission (2C). Recommendations specific to pediatric severe sepsis include: therapy with face mask oxygen, high flow nasal cannula oxygen, or nasopharyngeal continuous PEEP in the presence of respiratory distress and hypoxemia (2C), use of physical examination therapeutic endpoints such as capillary refill (2C); for septic shock associated with hypovolemia, the use of crystalloids or albumin to deliver a bolus of 20 mL/kg of crystalloids (or albumin equivalent) over 5-<em>1</em>0 min (2C); more common use of inotropes and vasodilators for low cardiac output septic shock associated with elevated systemic vascular resistance (2C); and use of hydrocortisone only in children with suspected or proven "absolute"' adrenal insufficiency (2C).

CONCLUSIONS

Strong agreement existed among a large cohort of international experts regarding many level <em>1</em> recommendations for the best care of patients with severe sepsis. Although a significant number of aspects of care have relatively weak support, evidence-based recommendations regarding the acute management of sepsis and septic shock are the foundation of improved outcomes for this important group of critically ill patients.

This article summarizes the new 20<em>1</em><em>1</em> report on dietary requirements for calcium and vitamin D from the Institute of Medicine (IOM). An IOM Committee charged with determining the population needs for these nutrients in North America conducted a comprehensive review of the evidence for both skeletal and extraskeletal outcomes. The Committee concluded that available scientific evidence supports a key role of calcium and vitamin D in skeletal health, consistent with a cause-and-effect relationship and providing a sound basis for determination of intake requirements. For extraskeletal outcomes, including cancer, cardiovascular disease, diabetes, and autoimmune disorders, the evidence was inconsistent, inconclusive as to causality, and insufficient to inform nutritional requirements. Randomized clinical trial evidence for extraskeletal outcomes was limited and generally uninformative. Based on bone health, Recommended Dietary Allowances (RDAs; covering requirements of ≥97.5% of the population) for calcium range from 700 to <em>1</em>300 mg/d for life-stage groups at least <em>1</em> yr of age. For vitamin D, RDAs of 600 IU/d for ages <em>1</em>-70 yr and 800 IU/d for ages 7<em>1</em> yr and older, corresponding to a serum 25-hydroxyvitamin D level of at least 20 ng/<em>ml</em> (50 nmol/liter), meet the requirements of at least 97.5% of the population. RDAs for vitamin D were derived based on conditions of minimal sun exposure due to wide variability in vitamin D synthesis from ultraviolet light and the risks of skin cancer. Higher values were not consistently associated with greater benefit, and for some outcomes U-shaped associations were observed, with risks at both low and high levels. The Committee concluded that the prevalence of vitamin D inadequacy in North America has been overestimated. Urgent research and clinical priorities were identified, including reassessment of laboratory ranges for 25-hydroxyvitamin D, to avoid problems of both undertreatment and overtreatment.

Vectors derived from human immunodeficiency virus (HIV) are highly efficient vehicles for in vivo gene delivery. However, their biosafety is of major concern. Here we exploit the complexity of the HIV genome to provide lentivirus vectors with novel biosafety features. In addition to the structural genes, HIV contains two regulatory genes, tat and rev, that are essential for HIV replication, and four accessory genes that encode critical virulence factors. We previously reported that the HIV type <em>1</em> accessory open reading frames are dispensable for efficient gene transduction by a lentivirus vector. We now demonstrate that the requirement for the tat gene can be offset by placing constitutive promoters upstream of the vector transcript. Vectors generated from constructs containing such a chimeric long terminal repeat (LTR) transduced neurons in vivo at very high efficiency, whether or not they were produced in the presence of Tat. When the rev gene was also deleted from the packaging construct, expression of gag and pol was strictly dependent on Rev complementation in trans. By the combined use of a separate nonoverlapping Rev expression plasmid and a 5' LTR chimeric transfer construct, we achieved optimal yields of vector of high transducing efficiency (up to <em>1</em>0(7) transducing units [TU]/<em>ml</em> and <em>1</em>0(4) TU/ng of p24). This third-generation lentivirus vector uses only a fractional set of HIV genes: gag, pol, and rev. Moreover, the HIV-derived constructs, and any recombinant between them, are contingent on upstream elements and trans complementation for expression and thus are nonfunctional outside of the vector producer cells. This split-genome, conditional packaging system is based on existing viral sequences and acts as a built-in device against the generation of productive recombinants. While the actual biosafety of the vector will ultimately be proven in vivo, the improved design presented here should facilitate testing of lentivirus vectors.

OBJECTIVE

There is a need for clearly defined and widely applicable clinical criteria for the selection of patients who may benefit from hepatic resection for metastatic colorectal cancer. Such criteria would also be useful for stratification of patients in clinical trials for this disease.

METHODS

Clinical, pathologic, and outcome data for <em>1</em>00<em>1</em> consecutive patients undergoing liver resection for metastatic colorectal cancer between July <em>1</em>985 and October <em>1</em>998 were examined. These resections included 237 trisegmentectomies, 394 lobectomies, and 370 resections encompassing less than a lobe. The surgical mortality rate was 2.8%.

RESULTS

The 5-year survival rate was 37%, and the <em>1</em>0-year survival rate was 22%. Seven factors were found to be significant and independent predictors of poor long-term outcome by multivariate analysis: positive margin (p = 0.004), extrahepatic disease (p = 0.003), node-positive primary (p = 0.02), disease-free interval from primary to metastases (<em>1</em>2 months (p = 0.03), number of hepatic tumors>><em>1</em> (p = 0.0004), largest hepatic tumor >5 cm (p = 0.0<em>1</em>), and carcinoembryonic antigen level >200 ng/<em>ml</em> (p = 0.0<em>1</em>). When the last five of these criteria were used in a preoperative scoring system, assigning one point for each criterion, the total score was highly predictive of outcome (p < 0.000<em>1</em>). No patient with a score of 5 was a long-term survivor.

CONCLUSIONS

Resection of hepatic colorectal metastases may produce long-term survival and cure. Long-term outcome can be predicted from five criteria that are readily available for all patients considered for resection. Patients with up to two criteria can have a favorable outcome. Patients with three, four, or five criteria should be considered for experimental adjuvant trials. Studies of preoperative staging techniques or of adjuvant therapies should consider using such a score for stratification of patients.

The inhibition of replicative DNA synthesis that follows DNA damage may be critical for avoiding genetic lesions that could contribute to cellular transformation. Exposure of <em>ML</em>-<em>1</em> myeloblastic leukemia cells to nonlethal doses of the DNA damaging agents, gamma-irradiation or actinomycin D, causes a transient inhibition of replicative DNA synthesis via both G<em>1</em> and G2 arrests. Levels of p53 protein in <em>ML</em>-<em>1</em> cells and in proliferating normal bone marrow myeloid progenitor cells increase and decrease in temporal association with the G<em>1</em> arrest. In contrast, the S-phase arrest of <em>ML</em>-<em>1</em> cells caused by exposure to the anti-metabolite, cytosine arabinoside, which does not directly damage DNA, is not associated with a significant change in p53 protein levels. Caffeine treatment blocks both the G<em>1</em> arrest and the induction of p53 protein after gamma-irradiation, thus suggesting that blocking the induction of p53 protein may contribute to the previously observed effects of caffeine on cell cycle changes after DNA damage. Unlike <em>ML</em>-<em>1</em> cells and normal bone marrow myeloid progenitor cells, hematopoietic cells that either lack p53 gene expression or overexpress a mutant form of the p53 gene do not exhibit a G<em>1</em> arrest after gamma-irradiation; however, the G2 arrest is unaffected by the status of the p53 gene. These results suggest a role for the wild-type p53 protein in the inhibition of DNA synthesis that follows DNA damage and thus suggest a new mechanism for how the loss of wild-type p53 might contribute to tumorigenesis.

Phosphatidylinositol (PtdIns) 3-kinase is an enzyme implicated in growth factor signal transduction by associating with receptor and nonreceptor tyrosine kinases, including the platelet-derived growth factor receptor. Inhibitors of PtdIns 3-kinase could potentially give a better understanding of the function and regulatory mechanisms of the enzyme. Quercetin, a naturally occurring bioflavinoid, was previously shown to inhibit PtdIns 3-kinase with an IC50 of <em>1</em>.3 microgram/<em>ml</em> (3.8 microM); inhibition appeared to be directed at the ATP-binding site of the kinase. Analogs of quercetin were investigated as PtdIns 3-kinase inhibitors, with the most potent ones exhibiting IC50 values in the range of <em>1</em>.7-8.4 micrograms/<em>ml</em>. In contrast, genistein, a potent tyrosine kinase inhibitor of the isoflavone class, did not inhibit PtdIns 3-kinase significantly (IC50>> 30 micrograms/<em>ml</em>). Since quercetin has also been shown to inhibit other PtdIns and protein kinases, other chromones were evaluated as inhibitors of PtdIns 3-kinase without affecting PtdIns 4-kinase or selected protein kinases. One such compound, 2-(4-morpholinyl)-8-phenyl-4H-<em>1</em>-benzopyran-4-one (also known as 2-(4-morpholinyl)-8-phenylchromone, LY294002), completely and specifically abolished PtdIns 3-kinase activity (IC50 = 0.43 microgram/<em>ml</em>; <em>1</em>.40 microM) but did not inhibit PtdIns 4-kinase or tested protein and lipid kinases. Analogs of LY294002 demonstrated a very selective structure-activity relationship, with slight changes in structure causing marked decreases in inhibition. LY294002 was shown to completely abolish PtdIns 3-kinase activity in fMet-Leu-Phe-stimulated human neutrophils, as well as inhibit proliferation of smooth muscle cells in cultured rabbit aortic segments. Since PtdIns 3-kinase appears to be centrally involved with growth factor signal transduction, the development of specific inhibitors against the kinase may be beneficial in the treatment of proliferative diseases as well as in elucidating the biological role of the kinase in cellular proliferation and growth factor response.

We revisit statistical tests for branches of evolutionary trees reconstructed upon molecular data. A new, fast, approximate likelihood-ratio test (aLRT) for branches is presented here as a competitive alternative to nonparametric bootstrap and Bayesian estimation of branch support. The aLRT is based on the idea of the conventional LRT, with the null hypothesis corresponding to the assumption that the inferred branch has length 0. We show that the LRT statistic is asymptotically distributed as a maximum of three random variables drawn from the chi(0)2 + chi(<em>1</em>)2 distribution. The new aLRT of interior branch uses this distribution for significance testing, but the test statistic is approximated in a slightly conservative but practical way as 2(l<em>1</em>- l2), i.e., double the difference between the maximum log-likelihood values corresponding to the best tree and the second best topological arrangement around the branch of interest. Such a test is fast because the log-likelihood value l2 is computed by optimizing only over the branch of interest and the four adjacent branches, whereas other parameters are fixed at their optimal values corresponding to the best <em>ML</em> tree. The performance of the new test was studied on simulated 4-, <em>1</em>2-, and <em>1</em>00-taxon data sets with sequences of different lengths. The aLRT is shown to be accurate, powerful, and robust to certain violations of model assumptions. The aLRT is implemented within the algorithm used by the recent fast maximum likelihood tree estimation program PHY<em>ML</em> (Guindon and Gascuel, 2003).

BACKGROUND

Observational data and non-human primate challenge studies suggest that cell-mediated immune responses might provide control of HIV replication. The Step Study directly assessed the efficacy of a cell-mediated immunity vaccine to protect against HIV-<em>1</em> infection or change in early plasma HIV-<em>1</em> levels.

METHODS

We undertook a double-blind, phase II, test-of-concept study at 34 sites in North America, the Caribbean, South America, and Australia. We randomly assigned 3000 HIV-<em>1</em>-seronegative participants by computer-generated assignments to receive three injections of MRKAd5 HIV-<em>1</em> gag/pol/nef vaccine (n=<em>1</em>494) or placebo (n=<em>1</em>506). Randomisation was prestratified by sex, adenovirus type 5 (Ad5) antibody titre at baseline, and study site. Primary objective was a reduction in HIV-<em>1</em> acquisition rates (tested every 6 months) or a decrease in HIV-<em>1</em> viral-load setpoint (early plasma HIV-<em>1</em> RNA measured 3 months after HIV-<em>1</em> diagnosis). Analyses were per protocol and modified intention to treat. The study was stopped early because it unexpectedly met the prespecified futility boundaries at the first interim analysis. This study is registered with ClinicalTrials.gov, number NCT00095576.

RESULTS

In a prespecified interim analysis in participants with baseline Ad5 antibody titre 200 or less, 24 (3%) of 74<em>1</em> vaccine recipients became HIV-<em>1</em> infected versus 2<em>1</em> (3%) of 762 placebo recipients (hazard ratio [HR] <em>1</em>.2 [95% CI 0.6-2.2]). All but one infection occurred in men. The corresponding geometric mean plasma HIV-<em>1</em> RNA was comparable in infected male vaccine and placebo recipients (4.6<em>1</em> vs 4.4<em>1</em> log(<em>1</em>0) copies per mL, one tailed p value for potential benefit 0.66). The vaccine elicited interferon-gamma ELISPOT responses in 75% (267) of the 25% random sample of all vaccine recipients (including both low and high Ad5 antibody titres) on whose specimens this testing was done (n=354). In exploratory analyses of all study volunteers, irrespective of baseline Ad5 antibody titre, the HR of HIV-<em>1</em> infection between vaccine and placebo recipients was higher in Ad5 seropositive men (HR 2.3 [95% CI <em>1</em>.2-4.3]) and uncircumcised men (3.8 [<em>1</em>.5-9.3]), but was not increased in Ad5 seronegative (<em>1</em>.0 [0.5-<em>1</em>.9]) or circumcised (<em>1</em>.0 [0.6-<em>1</em>.7]) men.

CONCLUSIONS

This cell-mediated immunity vaccine did not prevent HIV-<em>1</em> infection or reduce early viral level. Mechanisms for insufficient efficacy of the vaccine and the increased HIV-<em>1</em> infection rates in subgroups of vaccine recipients are being explored.

Anatomic and physiologic data are used to analyze the energy expenditure on different components of excitatory signaling in the grey matter of rodent brain. Action potentials and postsynaptic effects of glutamate are predicted to consume much of the energy (47% and 34%, respectively), with the resting potential consuming a smaller amount (<em>1</em>3%), and glutamate recycling using only 3%. Energy usage depends strongly on action potential rate--an increase in activity of <em>1</em> action potential/cortical neuron/s will raise oxygen consumption by <em>1</em>45 <em>mL</em>/<em>1</em>00 g grey matter/h. The energy expended on signaling is a large fraction of the total energy used by the brain; this favors the use of energy efficient neural codes and wiring patterns. Our estimates of energy usage predict the use of distributed codes, with <or=<em>1</em>5% of neurons simultaneously active, to reduce energy consumption and allow greater computing power from a fixed number of neurons. Functional magnetic resonance imaging signals are likely to be dominated by changes in energy usage associated with synaptic currents and action potential propagation.

The synthetic peptide T-20 (enfuvirtide) represents the first of a new class of antiretroviral compounds to demonstrate in vivo potency by targeting a step in viral entry. T-20 inhibits a conformational change in the human immunodeficiency virus type <em>1</em> (HIV-<em>1</em>) transmembrane glycoprotein (gp4<em>1</em>) that is required for fusion between HIV-<em>1</em> and target cell membranes. The initial phase I clinical trial of T-20 treatment for HIV-infected patients thus provided a unique opportunity to evaluate the emergence of resistant virus in vivo to this novel class of antiretroviral agents. All four patients who received an intermediate dose of T-20 (30 mg twice daily) had an initial decline in plasma viral load over the first <em>1</em>0 days but a rising trend by day <em>1</em>4, suggestive of selection for resistant virus. Plasma virus derived from patients enrolled in all dosage groups of the phase I T-20 trial was analyzed by population sequencing before and after treatment. While no mutations were found within a highly conserved 3-amino-acid sequence (GIV) known to be critical for fusion at baseline, after <em>1</em>4 days of therapy, virus from one patient in the 30-mg dose group (30-<em>1</em>) developed a mutation in this motif, specifically an aspartic acid (D) substitution for glycine (G) at position 36. Multiple env clones were derived from the plasma virus of all four patients in the 30-mg dosage group. Sequence analysis of 49 clones derived from the plasma of patient 30-<em>1</em> on day <em>1</em>4 revealed that 25 clones contained the G36D mutation, while 8 contained the V38A mutation. Dual mutations involving G36D and other residues within the HR<em>1</em> domain were also identified. In 5 of the 49 env clones, other mutations involving residues 32 (Q32R or Q32H) and 39 (Q39R) were found in combination with G36D. Cloned env sequences derived from the plasma virus of subject 30-3 also had single mutations in the GIV sequence (V38M and I37V) detectable following therapy with T-20. The plasma virus from subjects 30-2 and 30-4 did not contain changes within the GIV sequence. To analyze the biological resistance properties of these mutations, we developed a novel single-cycle HIV-<em>1</em> entry assay using JC53BL cells which express beta-galactosidase and luciferase under control of the HIV-<em>1</em> long terminal repeat. Full-length env clones were derived from the plasma virus of patients 30-<em>1</em> and 30-3 and used to generate pseudotyped virus stocks. The mean 50% inhibition concentrations (IC(50)s) for mutants G36D and V38A (patient 30-<em>1</em>) were 2.3 microg/<em>ml</em> and <em>1</em><em>1</em>.2 microg/<em>ml</em>, respectively, statistically significant increases of 9.<em>1</em>- and 45-fold, respectively, compared with those of wild-type Env. The IC(50) for the V38 M mutation (patient 30-3) was 7.6 microg/<em>ml</em>, an 8-fold increase compared with that of the wild type. The I37V mutation resulted in an IC(50) 3.2-fold greater than that of the wild type. Envs with double mutations (Q32R plus G36D and Q32H plus G36D) exhibited a level of resistance similar to that of G36D alone. These findings provide the first evidence for the rapid emergence of clinical resistance to a novel class of HIV-<em>1</em> entry inhibitors and may be relevant to future treatment strategies involving these agents.

The Schwartz formula was devised in the mid-<em>1</em>970s to estimate GFR in children. Recent data suggest that this formula currently overestimates GFR as measured by plasma disappearance of iohexol, likely a result of a change in methods used to measure creatinine. Here, we developed equations to estimate GFR using data from the baseline visits of 349 children (aged <em>1</em> to <em>1</em>6 yr) in the Chronic Kidney Disease in Children (CKiD) cohort. Median iohexol-GFR (iGFR) was 4<em>1</em>.3 <em>ml</em>/min per <em>1</em>.73 m(2) (interquartile range 32.0 to 5<em>1</em>.7), and median serum creatinine was <em>1</em>.3 mg/dl. We performed linear regression analyses assessing precision, goodness of fit, and accuracy to develop improvements in the GFR estimating formula, which was based on height, serum creatinine, cystatin C, blood urea nitrogen, and gender. The best equation was: GFR(<em>ml</em>/min per <em>1</em>.73 m(2))=39.<em>1</em>[height (m)/Scr (mg/dl)](0.5<em>1</em>6) x [<em>1</em>.8/cystatin C (mg/L)](0.294)[30/BUN (mg/dl)](0.<em>1</em>69)[<em>1</em>.099](male)[height (m)/<em>1</em>.4](0.<em>1</em>88). This formula yielded 87.7% of estimated GFR within 30% of the iGFR, and 45.6% within <em>1</em>0%. In a test set of <em>1</em>68 CKiD patients at <em>1</em> yr of follow-up, this formula compared favorably with previously published estimating equations for children. Furthermore, with height measured in cm, a bedside calculation of 0.4<em>1</em>3*(height/serum creatinine), provides a good approximation to the estimated GFR formula. Additional studies of children with higher GFR are needed to validate these formulas for use in screening all children for CKD.

OBJECTIVE

The purpose of this study was to determine the accuracy, precision, and linearity of the CellSearch system and evaluate the number of circulating tumor cells (CTCs) per 7.5 mL of blood in healthy subjects, patients with nonmalignant diseases, and patients with a variety of metastatic carcinomas.

METHODS

The CellSearch system was used to enumerate CTCs in 7.5 mL of blood. Blood samples spiked with cells from tumor cell lines were used to establish analytical accuracy, reproducibility, and linearity. Prevalence of CTCs was determined in blood from 199 patients with nonmalignant diseases, 964 patients with metastatic carcinomas, and 145 healthy donors.

RESULTS

Enumeration of spiked tumor cells was linear over the range of 5 to 1,142 cells, with an average recovery of>>/=85% at each spike level. Only 1 of the 344 (0.3%) healthy and nonmalignant disease subjects had>>/=2 CTCs per 7.5 mL of blood. In 2,183 blood samples from 964 metastatic carcinoma patients, CTCs ranged from 0 to 23,618 CTCs per 7.5 mL (mean, 60 +/- 693 CTCs per 7.5 mL), and 36% (781 of 2,183) of the specimens had>>/=2 CTCs. Detection of>>/=2 CTCs occurred at the following rates: 57% (107 of 188) of prostate cancers, 37% (489 of 1,316) of breast cancers, 37% (20 of 53) of ovarian cancers, 30% (99 of 333) of colorectal cancers, 20% (34 of 168) of lung cancers, and 26% (32 of 125) of other cancers.

CONCLUSIONS

The CellSearch system can be standardized across multiple laboratories and may be used to determine the clinical utility of CTCs. CTCs are extremely rare in healthy subjects and patients with nonmalignant diseases but present in various metastatic carcinomas with a wide range of frequencies.

BACKGROUND

Trials of endovascular therapy for ischemic stroke have produced variable results. We conducted this study to test whether more advanced imaging selection, recently developed devices, and earlier intervention improve outcomes.

METHODS

We rando<em>ml</em>y assigned patients with ischemic stroke who were receiving 0.9 mg of alteplase per kilogram of body weight less than 4.5 hours after the onset of ischemic stroke either to undergo endovascular thrombectomy with the Solitaire FR (Flow Restoration) stent retriever or to continue receiving alteplase alone. All the patients had occlusion of the internal carotid or middle cerebral artery and evidence of salvageable brain tissue and ischemic core of less than 70 <em>ml</em> on computed tomographic (CT) perfusion imaging. The coprimary outcomes were reperfusion at 24 hours and early neurologic improvement (≥8-point reduction on the National Institutes of Health Stroke Scale or a score of 0 or <em>1</em> at day 3). Secondary outcomes included the functional score on the modified Rankin scale at 90 days.

RESULTS

The trial was stopped early because of efficacy after 70 patients had undergone randomization (35 patients in each group). The percentage of ischemic territory that had undergone reperfusion at 24 hours was greater in the endovascular-therapy group than in the alteplase-only group (median, <em>1</em>00% vs. 37%; P<0.00<em>1</em>). Endovascular therapy, initiated at a median of 2<em>1</em>0 minutes after the onset of stroke, increased early neurologic improvement at 3 days (80% vs. 37%, P=0.002) and improved the functional outcome at 90 days, with more patients achieving functional independence (score of 0 to 2 on the modified Rankin scale, 7<em>1</em>% vs. 40%; P=0.0<em>1</em>). There were no significant differences in rates of death or symptomatic intracerebral hemorrhage.

CONCLUSIONS

In patients with ischemic stroke with a proximal cerebral arterial occlusion and salvageable tissue on CT perfusion imaging, early thrombectomy with the Solitaire FR stent retriever, as compared with alteplase alone, improved reperfusion, early neurologic recovery, and functional outcome. (Funded by the Australian National Health and Medical Research Council and others; EXTEND-IA ClinicalTrials.gov number, NCT0<em>1</em>492725, and Australian New Zealand Clinical Trials Registry number, ACTRN<em>1</em>26<em>1</em><em>1</em>000969965.).

BACKGROUND

Serum hepatitis B virus (HBV) DNA level is a marker of viral replication and efficacy of antiviral treatment in individuals with chronic hepatitis B.

OBJECTIVE

To evaluate the relationship between serum HBV DNA level and risk of hepatocellular carcinoma.

METHODS

Prospective cohort study of 3653 participants (aged 30-65 years), who were seropositive for the hepatitis B surface antigen and seronegative for antibodies against the hepatitis C virus, recruited to a community-based cancer screening program in Taiwan between <em>1</em>99<em>1</em> and <em>1</em>992.

METHODS

Incidence of hepatocellular carcinoma during follow-up examination and by data linkage with the national cancer registry and the death certification systems.

RESULTS

There were <em>1</em>64 incident cases of hepatocellular carcinoma and 346 deaths during a mean follow-up of <em>1</em><em>1</em>.4 years and 4<em>1</em>,779 person-years of follow-up. The incidence of hepatocellular carcinoma increased with serum HBV DNA level at study entry in a dose-response relationship ranging from <em>1</em>08 per <em>1</em>00,000 person-years for an HBV DNA level of less than 300 copies/<em>mL</em> to <em>1</em><em>1</em>52 per <em>1</em>00,000 person-years for an HBV DNA level of <em>1</em> million copies/<em>mL</em> or greater. The corresponding cumulative incidence rates of hepatocellular carcinoma were <em>1</em>.3% and <em>1</em>4.9%, respectively. The biological gradient of hepatocellular carcinoma by serum HBV DNA levels remained significant (P<.00<em>1</em>) after adjustment for sex, age, cigarette smoking, alcohol consumption, serostatus for the hepatitis B e antigen (HBeAg), serum alanine aminotransferase level, and liver cirrhosis at study entry. The dose-response relationship was most prominent for participants who were seronegative for HBeAg with normal serum alanine aminotransferase levels and no liver cirrhosis at study entry. Participants with persistent elevation of serum HBV DNA level during follow-up had the highest hepatocellular carcinoma risk.

CONCLUSIONS

Elevated serum HBV DNA level >> or =<em>1</em>0,000 copies/<em>mL</em>) is a strong risk predictor of hepatocellular carcinoma independent of HBeAg, serum alanine aminotransferase level, and liver cirrhosis.

<AbstractText>Coronavirus disease 20<em>1</em>9 (COVID-<em>1</em>9) has resulted in considerable morbidity and mortality worldwide since December 20<em>1</em>9. However, information on cardiac injury in patients affected by COVID-<em>1</em>9 is limited.</AbstractText><AbstractText>To explore the association between cardiac injury and mortality in patients with COVID-<em>1</em>9.</AbstractText><AbstractText>This cohort study was conducted from January 20, 2020, to February <em>1</em>0, 2020, in a single center at Renmin Hospital of Wuhan University, Wuhan, China; the final date of follow-up was February <em>1</em>5, 2020. All consecutive inpatients with laboratory-confirmed COVID-<em>1</em>9 were included in this study.</AbstractText><AbstractText>Clinical laboratory, radiological, and treatment data were collected and analyzed. Outcomes of patients with and without cardiac injury were compared. The association between cardiac injury and mortality was analyzed.</AbstractText><AbstractText>A total of 4<em>1</em>6 hospitalized patients with COVID-<em>1</em>9 were included in the final analysis; the median age was 64 years (range, 2<em>1</em>-95 years), and 2<em>1</em><em>1</em> (50.7%) were female. Common symptoms included fever (334 patients [80.3%]), cough (<em>1</em>44 [34.6%]), and shortness of breath (<em>1</em><em>1</em>7 [28.<em>1</em>%]). A total of 82 patients (<em>1</em>9.7%) had cardiac injury, and compared with patients without cardiac injury, these patients were older (median [range] age, 74 [34-95] vs 60 [2<em>1</em>-90] years; P < .00<em>1</em>); had more comorbidities (eg, hypertension in 49 of 82 [59.8%] vs 78 of 334 [23.4%]; P < .00<em>1</em>); had higher leukocyte counts (median [interquartile range (IQR)], 9400 [6900-<em>1</em>3 800] vs 5500 [4200-7400] cells/<em>μL</em>) and levels of C-reactive protein (median [IQR], <em>1</em>0.2 [6.4-<em>1</em>7.0] vs 3.7 [<em>1</em>.0-7.3] mg/dL), procalcitonin (median [IQR], 0.27 [0.<em>1</em>0-<em>1</em>.22] vs 0.06 [0.03-0.<em>1</em>0] ng/<em>mL</em>), creatinine kinase-myocardial band (median [IQR], 3.2 [<em>1</em>.8-6.2] vs 0.9 [0.6-<em>1</em>.3] ng/<em>mL</em>), myohemoglobin (median [IQR], <em>1</em>28 [68-305] vs 39 [27-65] μg/L), high-sensitivity troponin I (median [IQR], 0.<em>1</em>9 [0.08-<em>1</em>.<em>1</em>2] vs <0.006 [<0.006-0.009] μg/L), N-terminal pro-B-type natriuretic peptide (median [IQR], <em>1</em>689 [698-3327] vs <em>1</em>39 [5<em>1</em>-335] pg/<em>mL</em>), aspartate aminotransferase (median [IQR], 40 [27-60] vs 29 [2<em>1</em>-40] U/L), and creatinine (median [IQR], <em>1</em>.<em>1</em>5 [0.72-<em>1</em>.92] vs 0.64 [0.54-0.78] mg/dL); and had a higher proportion of multiple mottling and ground-glass opacity in radiographic findings (53 of 82 patients [64.6%] vs <em>1</em>5 of 334 patients [4.5%]). Greater proportions of patients with cardiac injury required noninvasive mechanical ventilation (38 of 82 [46.3%] vs <em>1</em>3 of 334 [3.9%]; P < .00<em>1</em>) or invasive mechanical ventilation (<em>1</em>8 of 82 [22.0%] vs <em>1</em>4 of 334 [4.2%]; P < .00<em>1</em>) than those without cardiac injury. Complications were more common in patients with cardiac injury than those without cardiac injury and included acute respiratory distress syndrome (48 of 82 [58.5%] vs 49 of 334 [<em>1</em>4.7%]; P < .00<em>1</em>), acute kidney injury (7 of 82 [8.5%] vs <em>1</em> of 334 [0.3%]; P < .00<em>1</em>), electrolyte disturbances (<em>1</em>3 of 82 [<em>1</em>5.9%] vs <em>1</em>7 of 334 [5.<em>1</em>%]; P = .003), hypoproteinemia (<em>1</em><em>1</em> of 82 [<em>1</em>3.4%] vs <em>1</em>6 of 334 [4.8%]; P = .0<em>1</em>), and coagulation disorders (6 of 82 [7.3%] vs 6 of 334 [<em>1</em>.8%]; P = .02). Patients with cardiac injury had higher mortality than those without cardiac injury (42 of 82 [5<em>1</em>.2%] vs <em>1</em>5 of 334 [4.5%]; P < .00<em>1</em>). In a Cox regression model, patients with vs those without cardiac injury were at a higher risk of death, both during the time from symptom onset (hazard ratio, 4.26 [95% CI, <em>1</em>.92-9.49]) and from admission to end point (hazard ratio, 3.4<em>1</em> [95% CI, <em>1</em>.62-7.<em>1</em>6]).</AbstractText><AbstractText>Cardiac injury is a common condition among hospitalized patients with COVID-<em>1</em>9 in Wuhan, China, and it is associated with higher risk of in-hospital mortality.</AbstractText>

Chronic kidney disease (CKD) is a worldwide public health problem, with adverse outcomes of kidney failure, cardiovascular disease (CVD), and premature death. A simple definition and classification of kidney disease is necessary for international development and implementation of clinical practice guidelines. Kidney Disease: Improving Global Outcomes (KDIGO) conducted a survey and sponsored a controversies conference to (<em>1</em>) provide a clear understanding to both the nephrology and nonnephrology communities of the evidence base for the definition and classification recommended by Kidney Disease Quality Outcome Initiative (K/DOQI), (2) develop global consensus for the adoption of a simple definition and classification system, and (3) identify a collaborative research agenda and plan that would improve the evidence base and facilitate implementation of the definition and classification of CKD. The K/DOQI definition and classification were accepted, with clarifications. CKD is defined as kidney damage or glomerular filtration rate (GFR) <60 <em>mL</em>/min/<em>1</em>.73 m(2) for 3 months or more, irrespective of cause. Kidney damage in many kidney diseases can be ascertained by the presence of albuminuria, defined as albumin-to-creatinine ratio >30 mg/g in two of three spot urine specimens. GFR can be estimated from calibrated serum creatinine and estimating equations, such as the Modification of Diet in Renal Disease (MDRD) Study equation or the Cockcroft-Gault formula. Kidney disease severity is classified into five stages according to the level of GFR. Kidney disease treatment by dialysis and transplantation should be noted. Simple, uniform classifications of CKD by cause and by risks for kidney disease progression and CVD should be developed.

In vitro studies have demonstrated antibacterial activity of essential oils (EOs) against Listeria monocytogenes, Salmonella typhimurium, Escherichia coli O<em>1</em>57:H7, Shigella dysenteria, Bacillus cereus and Staphylococcus aureus at levels between 0.2 and <em>1</em>0 microl <em>ml</em>(-<em>1</em>). Gram-negative organisms are slightly less susceptible than gram-positive bacteria. A number of EO components has been identified as effective antibacterials, e.g. carvacrol, thymol, eugenol, perillaldehyde, cinnamaldehyde and cinnamic acid, having minimum inhibitory concentrations (MICs) of 0.05-5 microl <em>ml</em>(-<em>1</em>) in vitro. A higher concentration is needed to achieve the same effect in foods. Studies with fresh meat, meat products, fish, milk, dairy products, vegetables, fruit and cooked rice have shown that the concentration needed to achieve a significant antibacterial effect is around 0.5-20 microl g(-<em>1</em>) in foods and about 0.<em>1</em>-<em>1</em>0 microl <em>ml</em>(-<em>1</em>) in solutions for washing fruit and vegetables. EOs comprise a large number of components and it is likely that their mode of action involves several targets in the bacterial cell. The hydrophobicity of EOs enables them to partition in the lipids of the cell membrane and mitochondria, rendering them permeable and leading to leakage of cell contents. Physical conditions that improve the action of EOs are low pH, low temperature and low oxygen levels. Synergism has been observed between carvacrol and its precursor p-cymene and between cinnamaldehyde and eugenol. Synergy between EO components and mild preservation methods has also been observed. Some EO components are legally registered flavourings in the EU and the USA. Undesirable organoleptic effects can be limited by careful selection of EOs according to the type of food.

BACKGROUND

Nintedanib (formerly known as BIBF <em>1</em><em>1</em>20) is an intracellular inhibitor that targets multiple tyrosine kinases. A phase 2 trial suggested that treatment with <em>1</em>50 mg of nintedanib twice daily reduced lung-function decline and acute exacerbations in patients with idiopathic pulmonary fibrosis.

METHODS

We conducted two replicate 52-week, randomized, double-blind, phase 3 trials (INPULSIS-<em>1</em> and INPULSIS-2) to evaluate the efficacy and safety of <em>1</em>50 mg of nintedanib twice daily as compared with placebo in patients with idiopathic pulmonary fibrosis. The primary end point was the annual rate of decline in forced vital capacity (FVC). Key secondary end points were the time to the first acute exacerbation and the change from baseline in the total score on the St. George's Respiratory Questionnaire, both assessed over a 52-week period.

RESULTS

A total of <em>1</em>066 patients were randomly assigned in a 3:2 ratio to receive nintedanib or placebo. The adjusted annual rate of change in FVC was -<em>1</em><em>1</em>4.7 ml with nintedanib versus -239.9 ml with placebo (difference, <em>1</em>25.3 ml; 95% confidence interval [CI], 77.7 to <em>1</em>72.8; P<0.00<em>1</em>) in INPULSIS-<em>1</em> and -<em>1</em><em>1</em>3.6 ml with nintedanib versus -207.3 ml with placebo (difference, 93.7 ml; 95% CI, 44.8 to <em>1</em>42.7; P<0.00<em>1</em>) in INPULSIS-2. In INPULSIS-<em>1</em>, there was no significant difference between the nintedanib and placebo groups in the time to the first acute exacerbation (hazard ratio with nintedanib, <em>1</em>.<em>1</em>5; 95% CI, 0.54 to 2.42; P=0.67); in INPULSIS-2, there was a significant benefit with nintedanib versus placebo (hazard ratio, 0.38; 95% CI, 0.<em>1</em>9 to 0.77; P=0.005). The most frequent adverse event in the nintedanib groups was diarrhea, with rates of 6<em>1</em>.5% and <em>1</em>8.6% in the nintedanib and placebo groups, respectively, in INPULSIS-<em>1</em> and 63.2% and <em>1</em>8.3% in the two groups, respectively, in INPULSIS-2.

CONCLUSIONS

In patients with idiopathic pulmonary fibrosis, nintedanib reduced the decline in FVC, which is consistent with a slowing of disease progression; nintedanib was frequently associated with diarrhea, which led to discontinuation of the study medication in less than 5% of patients. (Funded by Boehringer Ingelheim; INPULSIS-<em>1</em> and INPULSIS-2 ClinicalTrials.gov numbers, NCT0<em>1</em>335464 and NCT0<em>1</em>335477.).

BACKGROUND

Recently developed clinical practice guidelines and calibration of the Third National Health and Nutrition Examination Survey (NHANES III) serum creatinine assay provide a basis for estimating the prevalence and distribution of chronic kidney disease (CKD) in the United States using standardized criteria based on estimated glomerular filtration rate (GFR) and persistent albuminuria.

METHODS

A nationally representative sample of <em>1</em>5,625 noninstitutionalized adults aged 20 years and older from the NHANES III was analyzed. Kidney function (GFR), kidney damage (albuminuria), and stages of CKD (GFR and albuminuria) were estimated from calibrated serum creatinine level, spot urine albumin level, age, sex, and race. GFR was estimated using the simplified Modification of Diet in Renal Disease Study equation and compared with the Cockcroft-Gault equation for creatinine clearance (CCr).

RESULTS

The prevalence of CKD in the US adult population was <em>1</em><em>1</em>% (<em>1</em>9.2 million). By stage, an estimated 5.9 million individuals (3.3%) had stage <em>1</em> (persistent albuminuria with a normal GFR), 5.3 million (3.0%) had stage 2 (persistent albuminuria with a GFR of 60 to 89 <em>mL</em>/min/<em>1</em>.73 m(2)), 7.6 million (4.3%) had stage 3 (GFR, 30 to 59 <em>mL</em>/min/<em>1</em>.73 m(2)), 400,000 individuals (0.2%) had stage 4 (GFR, <em>1</em>5 to 29 <em>mL</em>/min/<em>1</em>.73 m(2)), and 300,000 individuals (0.2%) had stage 5, or kidney failure. Aside from hypertension and diabetes, age is a key predictor of CKD, and <em>1</em><em>1</em>% of individuals older than 65 years without hypertension or diabetes had stage 3 or worse CKD. Compared with GFR estimates, CCr estimates showed a steeper decline with age and were lower in non-Hispanic blacks.

CONCLUSIONS

CKD is common and warrants improved detection and classification using standardized criteria to improve outcomes. Am J Kidney Dis 4<em>1</em>:<em>1</em>-<em>1</em>2.