OBJECTIVE

The aim of this study was to evaluate the potential usefulness of whole-body (11)C-choline PET/CT in the re-staging of prostate cancer (PC) patients previously treated with radical prostatectomy (RP), who presented a mild increase of prostate-specific antigen (PSA) <1.5 ng/ml (early biochemical relapse) during follow-up (FU).

METHODS

We evaluated 102 consecutive patients (mean age = 68 years, range = 54-82 years) previously treated with RP and who presented during FU a mild increase of trigger PSA serum levels <1.5 ng/ml: mean 0.86 ± 0.40 ng/ml (range 0.2-1.5) and median 0.93 ng/ml (range 0.67-1.10). In this patient series (11)C-choline PET/CT was used as the first imaging examination at the time of the detection of a mild serum PSA increase <1.5 ng/ml. (11)C-Choline PET/CT was performed following standard procedures in our centre. At the time of PET/CT, 86 patients were not receiving any pharmacologic treatment, while 16 were under anti-androgenic therapy. Positive PET findings were validated by: (a) transrectal ultrasound (TRUS)-guided biopsy in cases of local recurrence, (b) surgical lymphadenectomy, (c) other imaging procedures or (d) FU lasting for at least 12 months. Univariate and multivariate analyses were used to evaluate the following variables: age, TNM staging, Gleason score, time from RP to the biochemical relapse, anti-androgen therapy at the time of (11)C-choline PET/CT scan, trigger PSA value and PSA kinetics, i.e. PSA doubling time (PSAdt) and PSA velocity (PSAvel), in order to assess the significant predictive factors related to the findings of a positive (11)C-choline PET/CT scan.

RESULTS

Overall, (11)C-choline PET/CT showed positive findings in 29 of 102 patients (28% of cases). In detail, (11)C-choline PET/CT detected: local relapse in 7 patients, bone metastases in 13 patients (4 single and 9 multiple) and lymph node metastases in 9 patients (6 single and 3 multiple). Positive PET findings were validated by: (a) TRUS-guided biopsy in 7 patients with local recurrence, (b) surgery and lymphadenectomy in 3 patients, (c) other targeted imaging procedures (MR or bone scan) in 5 patients and (d) clinical FU lasting a minimum of 12 months and including also a contrast-enhanced CT (CECT), an MR, a bone scan and a repeated (11)C-choline PET/CT in 14 patients. Age, time to biochemical relapse (TTR), initial T staging, Gleason score and trigger PSA were not statistically significant in predicting a positive (11)C-choline PET/CT scan both at univariate and multivariate analysis. Instead, PSA kinetics (PSAdt and PSAvel), N status and anti-androgenic therapy at the time of PET scan were statistically significant predictive factors at univariate analysis. Of note, only PSAdt and initial N status were found to be significant and independent predictive factors at multivariate analysis. The mean PSAdt in PET-positive patients was 4.34 months (SD 2.82) while in PET-negative patients it was 13.30 months (SD 9.75) (p = 0.0001). The optimal threshold for PSAdt established by receiver-operating characteristic (ROC) analysis was 7.25 months (AUC 0.85; 95% confidence interval 0.77-0.91) providing 93% sensitivity, 74% specificity, 60% positive predictive value and 96% negative predictive value.

CONCLUSIONS

In our study, (11)C-choline PET/CT was able to detect recurrent disease in 28% of the patients with mild biochemical relapse characterized by very low trigger PSA levels (PSA <1.5 ng/ml). Very interestingly (11)C-choline PET/CT detected distant unexpected metastases in 21% of the patients. At multivariate statistical analysis only PSAdt and node status were shown to be significant and independent predictive factors for positive (11)C-choline PET/CT. Therefore, (11)C-choline could be suggested to be performed early during initial biochemical relapse in patients presenting with fast PSA kinetics. The early detection of the site of recurrence could lead to a prompt instauration of the most appropriate treatment, i.e. local surgery or radiation treatment vs systemic treatment. In this view, one of the main advantages should be the avoidance of unnecessary local radiotherapy in those patients showing distant metastasis at (11)C-choline PET/CT.

A large variety of natural products have been described as anti-HIV agents, and for a portion thereof the target of interaction has been identified. Cyanovirin-N, a 11-kDa protein from Cyanobacterium (blue-green alga) irreversibly inactivates HIV and also aborts cell-to-cell fusion and transmission of HIV, due to its high-affinity interaction with gp120. Various sulfated polysaccharides extracted from seaweeds (i.e., Nothogenia fastigiata, Aghardhiella tenera) inhibit the virus adsorption process. Ingenol derivatives may inhibit virus adsorption at least in part through down-regulation of CD4 molecules on the host cells. Inhibition of virus adsorption by flavanoids such as (-)epicatechin and its 3-O-gallate has been attributed to an irreversible interaction with gp120 (although these compounds are also known as reverse transcriptase inhibitors). For the triterpene glycyrrhizin (extracted from the licorice root Glycyrrhiza radix) the mode of anti-HIV action may at least in part be attributed to interference with virus-cell binding. The mannose-specific plant lectins from Galanthus, Hippeastrum, Narcissus, Epipac tis helleborine, and Listera ovata, and the N-acetylgl ucosamine-specific lectin from Urtica dioica would primarily be targeted at the virus-cell fusion process. Various other natural products seem to qualify as HIV-cell fusion inhibitors: the siamycins [siamycin I (BMY-29304), siamycin II (RP 71955, BMY 29303), and NP-06 (FR901724)] which are tricyclic 21-amino-acid peptides isolated from Streptomyces spp that differ from one another only at position 4 or 17 (valine or isoleucine in each case); the betulinic acid derivative RPR 103611, and the peptides tachyplesin and polyphemusin which are highly abundant in hemocyte debris of the horseshoe crabs Tachypleus tridentatus and Limulus polyphemus, i.e., the 18-amino-acid peptide T22 from which T134 has been derived. Both T22 and T134 have been shown to block T-tropic X4 HIV-1 strains through a specific antagonism with the HIV corecept or CXCR4. A number of natural products have been reported to interact with the reverse transcriptase, i.e., baicalin, avarol, avarone, psychotrine, phloroglucinol derivatives, and, in particular, calanolides (from the tropical rainforest tree, Calophyllum lanigerum) and inophyllums (from the Malaysian tree, Calophyllum inophyllum). The natural marine substance illimaquinone would be targeted at the RNase H function of the reverse transcriptase. Curcumin (diferuloylmethane, from turmeric, the roots/rhizomes of Curcuma spp), dicaffeoylquinic and dicaffeoylt artaric acids, L-chicoric acid, and a number of fungal metabolites (equisetin, phomasetin, oteromycin, and integric acid) have all been proposed as HIV-1 integrase inhibitors. Yet, we have recently shown that L-c hicoric acid owes its anti-HIV activity to a specific interaction with the viral envelope gp120 rather than integrase. A number of compounds would be able to inhibit HIV-1 gene expression at the transcription level: the flavonoid chrysin (through inhibition of casein kinase II, the antibacter ial peptides melittin (from bee venom) and cecropin, and EM2487, a novel substance produced by Streptomyces. (ABSTRACT TRUNCATED)

OBJECTIVE

To compare the effects of short-term dietary supplementation with tomato juice, vitamin E, and vitamin C on susceptibility of LDL to oxidation and circulating levels of C-reactive protein (C-RP) and cell adhesion molecules in patients with type 2 diabetes.

METHODS

There were 57 patients with well-controlled type 2 diabetes aged <75 years treated with placebo for 4 weeks and then randomized to receive tomato juice (500 ml/day), vitamin E (800 U/day), vitamin C (500 mg/day), or continued placebo treatment for 4 weeks. Susceptibility of LDL to oxidation (lag time) and plasma concentrations of lycopene, vitamin E, vitamin C, C-RP, vascular cell adhesion molecule 1, and intercellular adhesion molecule 1 were measured at the beginning of the study, after the placebo phase, and at the end of the study.

RESULTS

Plasma lycopene levels increased nearly 3-fold (P = 0.001), and the lag time in isolated LDL oxidation by copper ions increased by 42% (P = 0.001) in patients during supplementation with tomato juice. The magnitude of this increase in lag time was comparable with the corresponding increase during supplementation with vitamin E (54%). Plasma C-RP levels decreased significantly (-49%, P = 0.004) in patients who received vitamin E. Circulating levels of cell adhesion molecules and plasma glucose did not change significantly during the study.

CONCLUSIONS

This study indicates that consumption of commercial tomato juice increases plasma lycopene levels and the intrinsic resistance of LDL to oxidation almost as effectively as supplementation with a high dose of vitamin E, which also decreases plasma levels of C-RP, a risk factor for myocardial infarction, in patients with diabetes. These findings may be relevant to strategies aimed at reducing risk of myocardial infarction in patients with diabetes.

Recent experimental studies (Pusch and Neher, 1988) and theoretical studies (Oliva et al., 1988) have found that the pipette tip is a significant barrier to diffusion in the whole cell patch clamp configuration. In this paper, we extend the theoretical analysis of fluxes between the pipette and cell to include transmembrane fluxes. The general conclusions are: (a) within the pipette, ion fluxes are driven primarily by diffusion rather than voltage gradients. (b) At steady state there is a concentration difference between the bulk pipette and intracellular solution that is described by delta c = jRp/Dp, where delta c = 1 mM for a flux, j = 1 fmol/s, through a pipette of resistance, Rp = 1 M omega, filled with a solution of resistivity, p = 100 omega --cm, given a solute diffusion coefficient, D = 10(-5) cm2/s. (c) The time to steady state is always accelerated by membrane transport, regardless of the direction of transport. We apply our analysis to the measurement of transport by the Na/K pump and Na/Ca exchanger in cells from the ventricles of mammalian heart. We find that the binding curve for intracellular Na+ to the Na/K pump will appear significantly less steep and more linear if one does not correct for the concentration difference between intracellular and pipette Na+. Similar shifts in the binding curve for extracellular Na+ to the Na/Ca exchanger can occur due to depletion of intracellular Ca(+)+ when the exchanger is stimulated. Lastly, in Appendix we analyze the effects of mobile and fixed intracellular buffers on the movement of Ca(+)+ between the pipette and cell. Fixed buffers greatly slow the time for equilibration of pipette and intracellular Ca(+)+. Mobile buffers act like a shuttle system, as they carry Ca(+)+ from pipette to cell then diffuse back when they are empty. Vigorous transport by the Na/Ca exchanger depletes mobile buffered calcium, thus stimulating diffusion from the pipette to match the rate of Ca(+)+ transport. Moreover, we find that binding of Ca(+)+ to the exchanger can be affected by the mobile buffer.

We studied the survival of cone photoreceptors following the degeneration of rods in the rd mouse. Cones were visualized by selective expression of green fluorescent protein (GFP) following transduction with an adeno-associated virus (AAV) vector. As previously reported, many cones survive after the initial degeneration of the rods. Soon after the initial degeneration, they lose their outer segments and all but a vestigial inner segment; and they partially retract or lose their axon and synaptic pedicle. However, they retain many fundamental features of the cone phenotype, and for many weeks show a polarized morphology indicative of substantial regrowth of processes. The cells retain their laminar position, forming a cell row just distal to a much thinned outer plexiform layer. The somata subsequently enlarge. Most of the cells extend bipolar processes, recreating the original bipolar morphology of a photoreceptor cell--though now turned on its side relative to the native position. The cells express short- or middle-wavelength opsins, recoverin and connexin36. One or more of the polarized processes could often be shown to contain synaptic ribbons, as visualized by antibodies against RIBEYE. The cones do not express protein kinase C alpha, Go alpha, ChX10 or calbindin, markers of bipolar or horizontal cells. The partially differentiated cone morphology persists for at least several months, after which the processes begin to retract and there is slow loss of the cells. Thus, during the time following the loss of their rod-dominated microenvironment, the cones achieve a semi-stable state in which much of their normal phenotype is preserved. Cone photoreceptors in retinas of human RP donors appear from their morphology to undergo a similar progression. The therapeutic window for rescue of cone photoreceptors may be longer than would have been thought.

1. Whole-cell patch-clamp recordings were made from dissociated guinea-pig nodose and trigeminal ganglion neurons in culture to study second messenger mechanisms of the hyperpolarization-activated current (Ih) modulation. 2. Prostaglandin E2 (PGE2) and forskolin modulate Ih in primary afferents by shifting the activation curve in the depolarizing direction and increasing the maximum amplitude. 3. The cAMP analogues, RP-cAMP-S (an inhibitor of protein kinase A (PKA)) and SP-cAMP-S (an activator of PKA), both shifted the activation curve of Ih to more depolarized potentials and occluded the effects of forskolin. These results suggest that Ih is modulated by a direct action of the cAMP analogues. 4. Superfusion of other cyclic nucleotide analogues (8-Br-cAMP, 8-(4-chlorophenylthio)-cAMP and 8-Br-cGMP) mimicked the actions of forskolin and PGE2, but dibutyryl cGMP, 5'-AMP and adenosine had no effect on Ih. 8-Br-cAMP and 8-Br-cGMP had similar concentration response profiles, suggesting that Ih has little nucleotide selectivity. 5. The inhibitor peptide (PKI), the catalytic subunit of PKA (C subunit) and phosphatase inhibitors (microcystin and okadaic acid) had no effect on forskolin modulation of Ih. 6. These results indicate that Ih is regulated by cyclic nucleotides in sensory neurons. Positive regulation of Ih by prostaglandins produced during inflammation may lead to depolarization and facilitation of repetitive activity, and thus contribute to sensitization to painful stimuli.

We have made use of the cell-free SV40 DNA replication system to identify and characterize cellular proteins required for efficient DNA synthesis. One such protein, replication protein C (RP-C), was shown to be involved with SV40 large T antigen in the early stages of viral DNA replication in vitro. We demonstrate here that RP-C is identical to the catalytic subunit of cellular protein phosphatase 2A (PP2Ac). The purified protein dephosphorylates specific phosphoamino acid residues in T antigen, consistent with the hypothesis that SV40 DNA replication is regulated by modulating the phosphorylation state of the viral initiator protein. We also show that purified RP-C/PP2Ac preferentially stimulates SV40 DNA replication in extracts from early G1 phase cells. This finding suggests that the activity of a cellular factor that influences the net phosphorylation state of T antigen is cell cycle dependent.

The ribosome, as a catalyst for protein synthesis, is universal and essential for all organisms. Here we describe the structure of the genes encoding human ribosomal proteins (RPs) and compare this class of genes among several eukaryotes. Using genomic and full-length cDNA sequences, we characterized 73 RP genes and found that (1) transcription starts at a C residue within a characteristic oligopyrimidine tract; (2) the promoter region is GC rich, but often has a TATA box or similar sequence element; (3) the genes are small (4.4 kb), but have as many as 5.6 exons on average; (4) the initiator ATG is in the first or second exon and is within plus minus 5 bp of the first intron boundaries in about half of cases; and (5) 5'- and 3'-UTRs are significantly smaller (42 bp and 56 bp, respectively) than the genome average. Comparison of RP genes from humans, Drosophila melanogaster, Caenorhabditis elegans, and Saccharomyces cerevisiae revealed the coding sequences to be highly conserved (63% homology on average), although gene size and the number of exons vary. The positions of the introns are also conserved among these species as follows: 44% of human introns are present at the same position in either D. melanogaster or C. elegans, suggesting RP genes are highly suitable for studying the evolution of introns.

The spontaneous nonenzymatic deamidation of glutaminyl and asparaginyl residues of peptides and proteins has been observed both in vitro and in vivo. Deamidation may change the structure and function of a peptide or protein, potentially resulting in decreased bioactivity, as well as alterations in pharmacokinetics and antigenicity of the protein pharmaceutical. Therefore, it is necessary to monitor the effect of storage and formulation conditions on deamidation of a protein drug candidate. Of particular interest is the investigation of in vivo deamidation mechanisms of protein drug candidates. Several methods are available to characterize the deamidation of peptides and proteins. We present here a LC/MS/MS method used to evaluate the deamidation of an antibody after in vivo administration. A humanized monoclonal IgG1 antibody (MAb) has several "hot spots" for spontaneous deamidation. One site, amino acid residue Asn55 located in the CDR2 region of the heavy chain, is of particular interest since deamidation at this site greatly decreases the binding activity. MAb was administered to cynomolgus monkeys by intravenous and subcutaneous routes. At various times after dosing, monkey serum was prepared and MAb captured by the immobilized antigen or a goat anti-human IgG Fcgamma antibody. The captured MAb was treated with trypsin followed by endoproteinase Glu-C. The digests were separated on RP-HPLC and analyzed by MS/MS on Q-Tof Global mass spectrometer. Using this method, we were able to determine the deamidation half-life of amino acid residue Asn55 in vivo and the ratio of the deamidated derivatives, i.e., isoAsp55 and Asp55. The method is rapid and sensitive with low-nanogram quantities of protein detected in the biological matrix.

Translational initiation of hepatitis C virus (HCV) genome RNA occurs via its highly structured 5' noncoding region called the internal ribosome entry site (IRES). Recent studies indicate that HCV IRES and 40 S ribosomal subunit form a stable binary complex that is believed to be important for the subsequent assembly of the 48 S initiation complex. Ribosomal protein (rp) S9 has been suggested as the prime candidate protein for binding of the HCV IRES to the 40 S subunit. RpS9 has a molecular mass of approximately 25 kDa in UV cross-linking experiments. In the present study, we examined the approximately 25-kDa proteins of the 40 S ribosome that form complexes with the HCV IRES upon UV cross-linking. Immunoprecipitation with specific antibodies against two 25-kDa 40 S proteins, rpS5 and rpS9, clearly identified rpS5 as the protein bound to the IRES. Thus, our results support rpS5 as the critical element in positioning the HCV RNA on the 40 S ribosomal subunit during translation initiation.

Recent evidence has suggested that cAMP plays a role as a second messenger in the decrease in nociceptive threshold (or hyperalgesia) produced by agents acting on primary afferent terminals. In support of this hypothesis we report that intradermal injection of a direct activator of adenyl cyclase, forskolin, produces a dose-dependent hyperalgesia in the rat. The duration of this hyperalgesia was prolonged by the phosphodiesterase inhibitors, isobutylmethylxanthine and rolipram. Forskolin hyperalgesia was antagonized by the Rp isomer of cyclic adenosine-3'5'-monophosphothioate, an analog of cAMP that prevents the phosphorylation of the cAMP protein kinase. The Rp isomer of cyclic adenosine-3'5'-monophosphothioate also inhibited the hyperalgesia induced by a membrane-permeable analogue of cAMP, 8-bromocyclic adenosine monophosphate, as well as the hyperalgesia induced by agents that are presumed to act directly on primary afferent nociceptors: prostaglandin E2, prostaglandin I2, (8R,15S)-dihydroxyicosa(5E-9,11,13Z)tetraenoic acid; and the adenosine A2-agonist 2-phenylaminoadenosine. Although the cAMP second messenger system contributes to primary afferent hyperalgesia, we found no evidence for a contribution of protein kinase C. Thus, hyperalgesia induced by prostaglandin E2, prostacyclin (prostaglandin I2), (8R,15S)-dihydroxyicosa(5E-9,11,13Z)tetraenoic acid, the adenosine A2-agonist 2-phenylaminoadenosine, 8-bromocyclic adenosine monophosphate and the direct activator of adenyl cyclase, forskolin, were not significantly attenuated by the selective inhibition of protein kinase C by the 19-31 fragment of protein kinase C. Two other inhibitors of protein kinase C, sphingosine and staurosporine, also failed to attenuate prostaglandin E2-induced hyperalgesia.

Ku is a heterodimeric protein composed of approximately 70- and approximately 80-kDa subunits (Ku70 and Ku80) originally identified as an autoantigen recognized by the sera of patients with autoimmune diseases. Ku has high binding affinity for DNA ends and that is why originally it was known as a DNA end binding protein, but now it is known to also bind the DNA structure at nicks, gaps, hairpins, as well as the ends of telomeres. It has been reported also to bind with sequence specificity to DNA and with weak affinity to RNA. Ku is an abundant nuclear protein and is present in vertebrates, insects, yeast, and worms. Ku contains ssDNA-dependent ATPase and ATP-dependent DNA helicase activities. It is the regulatory subunit of the DNA-dependent protein kinase that phosphorylates many proteins, including SV-40 large T antigen, p53, RNA-polymerase II, RP-A, topoisomerases, hsp90, and many transcription factors such as c-Jun, c-Fos, oct-1, sp-1, c-Myc, TFIID, and many more. It seems to be a multifunctional protein that has been implicated to be involved directly or indirectly in many important cellular metabolic processes such as DNA double-strand break repair, V(D)J recombination of immunoglobulins and T-cell receptor genes, immunoglobulin isotype switching, DNA replication, transcription regulation, regulation of heat shock-induced responses, regulation of the precise structure of telomeric termini, and it also plays a novel role in G2 and M phases of the cell cycle. The mechanism underlying the regulation of all the diverse functions of Ku is still obscure.

We report a new quantitative metabolome profiling technique based on differential (12)C-/(13)C-isotope dansylation labeling of metabolites, fast liquid chromatography (LC) separation and electrospray ionization Fourier-transform ion cyclotron resonance mass spectrometry (ESI-FTICR MS) detection. An isotope reagent, (13)C-dansyl chloride, can be readily synthesized. This reagent, along with (12)C-dansyl chloride, provides a simple and robust means of labeling metabolites containing primary amine, secondary amine, or phenolic hydroxyl group(s). It is shown that dansylation labeling offers 1-3 orders of magnitude ESI signal enhancement over the underivatized counterparts. Dansylation alters the chromatographic behaviors of polar and ionic metabolites normally not retainable on a reversed phase (RP) column to an extent that they can be retained and separated by RPLC with high efficiency. There is no isotopic effect on RPLC separation of the differential isotope labeled metabolites, and (12)C-/(13)C-labeled isoforms of metabolites are coeluted and detected by MS for precise and accurate quantification and confident metabolite identification. It is demonstrated that, in the analysis of 20 amino acids, a linear response of over 2 orders of magnitude is achieved for relative metabolite quantification with an average relative standard deviation (RSD) of about 5.3% from replicate experiments. A dansylation standard compound library consisting of 121 known amines and phenols has been constructed and is proven to be useful for absolute metabolite quantification and MS-based metabolite identification in biological samples. As an example, the absolute concentrations of 93 metabolites, ranging from 30 nM to 2510 microM, can be determined from a pooled sample of human urines collected in 5 consecutive days labeled with (12)C-dansylation and spiked with the 121 (13)C-dansylated standards. Relative concentration variations of these metabolites in individual urine samples can also be monitored by mixing the (13)C-dansylated pooled urine sample with the (12)C-dansylated individual sample. With a 12 min fast LC separation combined with FTICR MS, 672 metabolites were detected in a human urine sample with each metabolite peak having a signal-to-noise ratio of greater than 20; the identities of most of the metabolites remain to be determined. This work illustrates that dansylation labeling and fast LC/FTICR MS can be a powerful technique for quantitative profiling of at least 672 metabolites in urine samples in 12 min.

The primary reservoir for hepatitis C virus (HCV) replication is believed to be hepatocytes, which are highly polarized with tight junctions (TJ) separating their basolateral and apical domains. HepG2 cells develop polarity over time, resulting in the formation and remodeling of bile canalicular (BC) structures. HepG2 cells expressing CD81 provide a model system to study the effects of hepatic polarity on HCV infection. We found an inverse association between HepG2-CD81 polarization and HCV pseudoparticle entry. As HepG2 cells polarize, discrete pools of claudin-1 (CLDN1) at the TJ and basal/lateral membranes develop, consistent with the pattern of receptor staining observed in liver tissue. The TJ and nonjunctional pools of CLDN1 show an altered association with CD81 and localization in response to the PKA antagonist Rp-8-Br-cyclic AMPs (cAMPs). Rp-8-Br-cAMPs reduced CLDN1 expression at the basal membrane and inhibited HCV infection, supporting a model where the nonjunctional pools of CLDN1 have a role in HCV entry. Treatment of HepG2 cells with proinflammatory cytokines, tumor necrosis factor alpha and gamma interferon, perturbed TJ integrity but had minimal effect(s) on cellular polarity and HCV infection, suggesting that TJ integrity does not limit HCV entry into polarized HepG2 cells. In contrast, activation of PKC with phorbol ester reduced TJ integrity, ablated HepG2 polarity, and stimulated HCV entry. Overall, these data show that complex hepatocyte-like polarity alters CLDN1 localization and limits HCV entry, suggesting that agents which disrupt hepatocyte polarity may promote HCV infection and transmission within the liver.

Background: Pediatric SARS-CoV-2 infection can be complicated by a dangerous hyperinflammatory condition termed multisystem inflammatory syndrome in children (MIS-C). The clinical and immunologic spectrum of MIS-C and its relationship to other inflammatory conditions of childhood have not been studied in detail.

Methods: We retrospectively studied confirmed cases of MIS-C at our institution from March to June 2020. The clinical characteristics, laboratory studies and treatment response were collected. Data were compared with historic cohorts of Kawasaki disease (KD) and macrophage activation syndrome (MAS).

Results: Twenty-eight patients fulfilled the case definition of MIS-C. Median age at presentation was 9 years (range 1 month to 17 years); 50% of patients had pre-existing conditions. All patients had laboratory confirmation of SARS-CoV-2 infection. Seventeen patients (61%) required intensive care, including 7 patients (25%) requiring inotrope support. Seven patients (25%) met criteria for complete or incomplete KD and coronary abnormalities were found in 6 cases. Lymphopenia, thrombocytopenia, and elevation in inflammatory markers, D-dimer, B-type natriuretic peptide, IL-6 and IL-10 levels were common but not ubiquitous. Cytopenias distinguished MIS-C from KD and the degree of hyperferritinemia and pattern of cytokine production differed between MIS-C and MAS. Immunomodulatory therapy given to MIS-C patients included IVIG (71%), corticosteroids (61%) and anakinra (18%). Clinical and laboratory improvement were observed in all cases, including 6 cases that did not require immunomodulatory therapy. No mortality was recorded in this cohort.

Conclusion: MIS-C encompasses a broad phenotypic spectrum with clinical and laboratory features distinct from Kawasaki disease and macrophage activation syndrome.

Funding: This work was supported by the National Institute of Health / National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS) K08-AR074562 (PYL), K08-AR AR073339 (LAH), R01-AR065538, R01-AR073201 and P30-AR070253 (PAN); National Institute of Allergy and Infectious Diseases 5T32AI007512-34 (JL, JR, TB, AAN and RWN); Rheumatology Research Foundation Investigator Awards (PYL and LAH) and Medical Education Award (JSH); Boston Children's Hospital Faculty Career Development Awards (PYL and LAH), the McCance Family Foundation (JWN), and the Samara Jan Turkel Center (JC, RPS, MBS).

Keywords: COVID-19; Clinical practice; Immunology.

BACKGROUND

The magnitude of surgical trauma after laparoscopic and open colonic resection was evaluated by examining postoperative serum values of interleukin-6 (IL-6), IL-10, C-reactive protein (CRP), and granulocyte elastase (GE) for further evidence of the benefit realized with minimally invasive approaches in colonic surgery.

METHODS

Altogether, 42 patients with Crohn's disease (n = 20) or colon carcinomas/adenomas (n = 22) were matched by age, gender, body mass index (BMI), and Crohn's Disease Activity Index for either a laparoscopic (n = 21) or an open colonic resection (n = 21). In both groups the postoperative serum levels of IL-6, IL-10, C-RP, and granulocyte elastase were determined, as indicators of surgical stress.

RESULTS

Laparoscopic and open colonic resection caused a significant increase in serum IL-6, IL-10, CRP, and granulocyte elastase levels. The comparison between laparoscopic and open colonic resections, however, showed significantly lower serum IL-6, IL-10, CRP, and granulocyte elastase levels after laparoscopic colonic resection, which was most evident for IL-6 and granulocyte elastase.

CONCLUSIONS

Our study demonstrated that IL-6 and granulocyte elastase may be appropriated particularly to monitor surgical stress. By using these parameters, we found a significant reduction in surgical trauma after laparoscopic surgery, was compared with the open procedure. This supports the clinical findings of a clear benefit for patients undergoing laparoscopic colonic surgery.

Inherited retinal degenerations, including retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA), comprise a group of disorders showing high genetic and allelic heterogeneity. The determination of a full catalog of genes that can, when mutated, cause human retinal disease is a powerful means to understand the molecular physiology and pathology of the human retina. As more genes are found, remaining ones are likely to be rarer and/or unexpected candidates. Here, we identify a family in which all known RP/LCA-related genes are unlikely to be associated with their disorder. A combination of homozygosity mapping and exome sequencing identifies a homozygous nonsense mutation, c.496C)T (p.Arg166X), in a gene, KCNJ13, encoding a potassium channel subunit Kir7.1. A screen of a further 333 unrelated individuals with recessive retinal degeneration identified an additional proband, homozygous for a missense mutation, c.722T>C (p.Leu241Pro), in the same gene. The three affected members of the two families have been diagnosed with LCA. All have a distinct and unusual retinal appearance and a similar early onset of visual loss, suggesting both impaired retinal development and progressive retinal degeneration, involving both rod and cone pathways. Examination of heterozygotes revealed no ocular disease. This finding implicates Kir7.1 as having an important role in human retinal development and maintenance. This disorder adds to a small diverse group of diseases consequent upon loss or reduced function of inwardly rectifying potassium channels affecting various organs. The distinct retinal phenotype that results from biallelic mutations in KCNJ13 should facilitate the molecular diagnosis in further families.

Monospecific antibodies have been prepared against cytochrome ccapsulata, and against cytochrome c' from Rps. capsulata. These antibodies precipitated their respective antigens, but did not cross react with a wide range of procaryotic or eucaryotic cytochromes, or with other bacterial proteins. The cytochromes produced during aerobic growth were immunologically indistinguishable from those produced during photosynthetic growth. Cytochrome ccated in vivo in the periplasmic space between the cell was and the cell membrane, and when chromatophores are prepared from whole cells the cytochrome becomes trapped inside these vesicles. The implications of these results to energy coupling in the photosynthetic bacteria are discussed.

Prp8 protein (Prp8p) is a highly conserved pre-mRNA splicing factor and a component of spliceosomal U5 small nuclear ribonucleoproteins (snRNPs). Although it is ubiquitously expressed, mutations in the C terminus of human Prp8p cause the retina-specific disease retinitis pigmentosa (RP). The biogenesis of U5 snRNPs is poorly characterized. We present evidence for a cytoplasmic precursor U5 snRNP in yeast that lacks the mature U5 snRNP component Brr2p and depends on a nuclear localization signal in Prp8p for its efficient nuclear import. The association of Brr2p with the U5 snRNP occurs within the nucleus. RP mutations in Prp8p in yeast result in nuclear accumulation of the precursor U5 snRNP, apparently as a consequence of disrupting the interaction of Prp8p with Brr2p. We therefore propose a novel assembly pathway for U5 snRNP complexes that is disrupted by mutations that cause human RP.

BACKGROUND

Studies of gene expression in post mortem human brain can contribute to understanding of the pathophysiology of neurodegenerative diseases, including Alzheimer's disease (AD), Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Quantitative real-time PCR (RT qPCR) is often used to analyse gene expression. The validity of results obtained using RT qPCR is reliant on accurate data normalization. Reference genes are generally used to normalize RT qPCR data. Given that expression of some commonly used reference genes is altered in certain conditions, this study aimed to establish which reference genes were stably expressed in post mortem brain tissue from individuals with AD, PD or DLB.

RESULTS

The present study investigated the expression stability of 8 candidate reference genes, (ubiquitin C [UBC], tyrosine-3-monooxygenase [YWHAZ], RNA polymerase II polypeptide [RP II], hydroxymethylbilane synthase [HMBS], TATA box binding protein [TBP], beta-2-microglobulin [B2M], glyceraldehyde-3-phosphate dehydrogenase [GAPDH], and succinate dehydrogenase complex-subunit A, [SDHA]) in cerebellum and medial temporal gyrus of 6 AD, 6 PD, 6 DLB subjects, along with 5 matched controls using RT qPCR (TaqMan(R) Gene Expression Assays). Gene expression stability was analysed using geNorm to rank the candidate genes in order of decreasing stability in each disease group. The optimal number of genes recommended for accurate data normalization in each disease state was determined by pairwise variation analysis.

CONCLUSIONS

This study identified validated sets of mRNAs which would be appropriate for the normalization of RT qPCR data when studying gene expression in brain tissue of AD, PD, DLB and control subjects.

Yeast 25S rRNA was reported to contain a single cytosine methylation (m(5)<em>C</em>). In the present study using a combination of <em>RP</em>-HPL<em>C</em>, mung bean nuclease assay and rRNA mutagenesis, we discovered that instead of one, yeast contains two m(5)<em>C</em> residues at position 2278 and 2870. Furthermore, we identified and characterized two putative methyltransferases, Rcm1 and Nop2 to be responsible for these two cytosine methylations, respectively. Both proteins are highly conserved, which correlates with the presence of two m(5)<em>C</em> residues at identical positions in higher eukaryotes, including humans. The human homolog of yeast Nop2, p120 has been discovered to be upregulated in various cancer tissues, whereas the human homolog of Rcm1, NSUN5 is completely deleted in the William's-Beuren Syndrome. The substrates and function of both human homologs remained unknown. In the present study, we also provide insights into the significance of these two m(5)<em>C</em> residues. The loss of m(5)<em>C</em>2278 results in anisomycin hypersensitivity, whereas the loss of m(5)<em>C</em>2870 affects ribosome synthesis and processing. Establishing the locations and enzymes in yeast will not only help identifying the function of their homologs in higher organisms, but will also enable understanding the role of these modifications in ribosome function and architecture.

Two distinct pathways for completion of base excision repair (BER) have been discovered in eukaryotes: the DNA polymerase beta (Pol beta)-dependent short-patch pathway that involves the replacement of a single nucleotide and the long-patch pathway that entails the resynthesis of 2-6 nucleotides and requires PCNA. We have used cell extracts from Pol beta-deleted mouse fibroblasts to separate subfractions containing either Pol delta or Pol epsilon. These fractions were then tested for their ability to perform both short- and long-patch BER in an in vitro repair assay, using a circular DNA template, containing a single abasic site at a defined position. Remarkably, both Pol delta and Pol epsilon were able to replace a single nucleotide at the lesion site, but the repair reaction is delayed compared to single nucleotide replacement by Pol beta. Furthermore, our observations indicated, that either Pol delta and/or Pol epsilon participate in the long-patch BER. PCNA and RF-C, but not RP-A are required for this process. Our data show for the first time that Pol delta and/or Pol epsilon are directly involved in the long-patch BER of abasic sites and might function as back-up system for Pol beta in one-gap filling reactions.

Two second messenger pathways, one that uses the cAMP-dependent protein kinase A (PKA), the other that uses protein kinase C (PKC), have been found to contribute to the short-term presynaptic facilitation of the connections between the sensory neurons in Aplysia and their target cells, the interneurons and motor neurons of the gill-withdrawal reflex. To study their relative contributions as a function of the previous history of the neuron's activity, we have examined the effects of inhibiting PKA (using Rp-cAMPS) and PKC (using H7) on the short-term facilitation of spontaneous release as well as of the evoked release induced by serotonin at nondepressed, partially depressed, and highly depressed synapses. Our results suggest that whereas activation of PKA is sufficient to trigger the facilitation of nondepressed synapses, activation of both PKA and PKC is required to facilitate depressed synapses, with the contribution of PKC becoming progressively more important as synaptic transmission becomes more depressed.

OBJECTIVE

To describe the structural changes in the transition zone from relatively healthy retinal regions to severely affected regions in patients with retinitis pigmentosa (RP) using frequency domain optical coherence tomography (fdOCT).

METHODS

FdOCT line scans of the horizontal meridian were obtained from one eye of 13 patients with RP and 30 control subjects. The patients had normal or near normal foveal sensitivities and visual field diameters ≥10°. Using a computer-aided manual segmentation procedure, the locations at which the outer segment (OS) and outer nuclear layer plus outer plexiform layer (ONL+) thicknesses fell below the 95% confidence interval of the controls were measured, as were the locations at which the OS layer disappeared and the locations at which the ONL+ was reduced to an asymptotically small thickness.

RESULTS

The progression from healthy to severely affected regions followed a common pattern in most patients. Region A, the central region including the foveal center, had normal OS and ONL+ thickness. Region B had abnormal OS but normal ONL+ thickness. Region C had abnormal but measurable OS and ONL+ thicknesses. In Region D, the OS layer disappeared, as did the IS/OS line, and the ONL+ thickness decreased further. In Region E, the ONL+ reached an asymptotic thickness.

CONCLUSIONS

The structural changes in the transition zone followed an orderly progression from a thinning of the OS layer, to a thinning of the ONL+, to a loss of the OS layer, to an ONL+ reduced to an asymptotically small level.