SMC5/6 Promotes Replication Fork Stability via Negative Regulation of the COP9 Signalosome.

It is widely accepted that DNA replication fork stalling is a common occurrence during cell proliferation, but there are robust mechanisms to alleviate this and ensure DNA replication is completed prior to chromosome segregation. The SMC5/6 complex has consistently been implicated in the maintenance of replication fork integrity. However, the essential role of the SMC5/6 complex during DNA replication in mammalian cells has not been elucidated. In this study, we investigate the molecular consequences of SMC5/6 loss at the replication fork in mouse embryonic stem cells (mESCs), employing the auxin-inducible degron (AID) system to deplete SMC5 acutely and reversibly in the defined cellular contexts of replication fork stall and restart. In SMC5-depleted cells, we identify a defect in the restart of stalled replication forks, underpinned by excess MRE11-mediated fork resection and a perturbed localization of fork protection factors to the stalled fork. Previously, we demonstrated a physical and functional interaction of SMC5/6 with the COP9 signalosome (CSN), a cullin deneddylase that enzymatically regulates cullin ring ligase (CRL) activity. Employing a combination of DNA fiber techniques, the AID system, small-molecule inhibition assays, and immunofluorescence microscopy analyses, we show that SMC5/6 promotes the localization of fork protection factors to stalled replication forks by negatively modulating the COP9 signalosome (CSN). We propose that the SMC5/6-mediated modulation of the CSN ensures that CRL activity and their roles in DNA replication fork stabilization are maintained to allow for efficient replication fork restart when a replication fork stall is alleviated.

Australian consumers' attitudes to packaged fresh meat products with added microbial bioprotective cultures for shelf-life extension.

The application of food-grade microbial cultures to fresh meat products is a promising natural approach for meat shelf-life extension. However, before its adoption into commercial practice, it is essential to understand consumers' attitudes to this approach and the resulting marketed products. This study investigated Australian consumers' willingness to purchase and consume packaged fresh meat products with added microbial cultures for shelf-life extension. A national online survey of over 800 respondents was conducted. Results indicated that most Australian consumers would be willing to buy and eat such products, with 17.8% of respondents less likely to buy and 11.1% unwilling to eat these products. Respondents' purchasing and consumption decisions were influenced by demographic factors, their food and meat shopping and consumption behaviors, and the value, taste, and type of the meat product. Consumer acceptance may be improved by increasing their awareness of the potential use of microbial cultures as natural antimicrobials for food shelf-life extension.

SMC5 Plays Independent Roles in Congenital Heart Disease and Neurodevelopmental Disability.

Up to 50% of patients with severe congenital heart disease (CHD) develop life-altering neurodevelopmental disability (NDD). It has been presumed that NDD arises in CHD cases because of hypoxia before, during, or after cardiac surgery. Recent studies detected an enrichment in de novo mutations in CHD and NDD, as well as significant overlap between CHD and NDD candidate genes. However, there is limited evidence demonstrating that genes causing CHD can produce NDD independent of hypoxia. A patient with hypoplastic left heart syndrome and gross motor delay presented with a de novo mutation in SMC5. Modeling mutation of smc5 in Xenopus tropicalis embryos resulted in reduced heart size, decreased brain length, and disrupted pax6 patterning. To evaluate the cardiac development, we induced the conditional knockout (cKO) of Smc5 in mouse cardiomyocytes, which led to the depletion of mature cardiomyocytes and abnormal contractility. To test a role for Smc5 specifically in the brain, we induced cKO in the mouse central nervous system, which resulted in decreased brain volume, and diminished connectivity between areas related to motor function but did not affect vascular or brain ventricular volume. We propose that genetic factors, rather than hypoxia alone, can contribute when NDD and CHD cases occur concurrently.

Predicting functional effect of missense variants using graph attention neural networks.

Accurate prediction of damaging missense variants is critically important for interpreting a genome sequence. Although many methods have been developed, their performance has been limited. Recent advances in machine learning and the availability of large-scale population genomic sequencing data provide new opportunities to considerably improve computational predictions. Here we describe the graphical missense variant pathogenicity predictor (gMVP), a new method based on graph attention neural networks. Its main component is a graph with nodes that capture predictive features of amino acids and edges weighted by co-evolution strength, enabling effective pooling of information from the local protein context and functionally correlated distal positions. Evaluation of deep mutational scan data shows that gMVP outperforms other published methods in identifying damaging variants in TP53, PTEN, BRCA1 and MSH2. Furthermore, it achieves the best separation of de novo missense variants in neuro developmental disorder cases from those in controls. Finally, the model supports transfer learning to optimize gain- and loss-of-function predictions in sodium and calcium channels. In summary, we demonstrate that gMVP can improve interpretation of missense variants in clinical testing and genetic studies.

Microbial biopreservatives for controlling the spoilage of beef and lamb meat: their application and effects on meat quality.

Biopreservation is a recognized natural method for controlling the growth of undesirable bacteria on fresh meat. It offers the potential to inhibit spoilage bacteria and extend meat shelf-life, but this aspect has been much less studied compared to using the approach to target pathogenic bacteria. This review provides comprehensive information on the application of biopreservatives of microbial origin, mainly bacteriocins and protective cultures, in relation to bacterial spoilage of beef and lamb meat. The sensory effect of these biopreservatives, an aspect that often receives less attention in microbiological studies, is also reviewed. Microbial biopreservatives were found to be able to retard the growth of the major meat spoilage bacteria, Brochothrix thermosphacta, Pseudomonas spp., and Enterobacteriaceae. Their addition did not have any discernible negative impact on the sensory properties of meat, whether assessed by human sensory panels or instrumental and chemical analyses. Although results are promising, the concept of biopreservation for controlling spoilage bacteria on fresh meat is still in its infancy. Studies in this area are still lacking, especially for lamb. Biopreservatives need more testing under conditions representative of commercial meat production, along with studies of any possible sensory effects, in order to validate their potential for large-scale industrial applications.

Evaluation of the potential of protective cultures to extend the microbial shelf-life of chilled lamb meat.

The use of protective cultures to inhibit spoilage bacteria is a promising natural preservation technique to extend the shelf-life of fresh meat. This study evaluated the effectiveness of six food-grade protective cultures (containing different combinations of Lactobacillus sakei, Pediococcus pentosaceus, Staphylococcus xylosus, and Staphylococcus carnosus) on naturally contaminated chill-stored (4 °C) lamb meat in different packaging systems. Only slight reductions of common meat spoilage bacteria Brochothrix thermosphacta, Pseudomonas spp., and Enterobacteriaceae were observed in culture-treated samples stored in modified atmosphere packaging (80% O2:20% CO2). Greater inhibitory effects were found in vacuum-packed lamb, with mixed cultures containing either L. sakei, S. carnosus, and S. xylosus or S. carnosus and L. sakei causing the most significant reductions. Protective cultures did not adversely affect meat color or pH. This study demonstrated the potential of protective cultures comprising lactic acid bacteria and coagulase-negative staphylococci in controlling microbial spoilage of lamb and, by inference, other types of meat as a natural solution for shelf-life extension.

SMC5/6 is required for replication fork stability and faithful chromosome segregation during neurogenesis.

Mutations of SMC5/6 components cause developmental defects, including primary microcephaly. To model neurodevelopmental defects, we engineered a mouse wherein Smc5 is conditionally knocked out (cKO) in the developing neocortex. Smc5 cKO mice exhibited neurodevelopmental defects due to neural progenitor cell (NPC) apoptosis, which led to reduction in cortical layer neurons. Smc5 cKO NPCs formed DNA bridges during mitosis and underwent chromosome missegregation. SMC5/6 depletion triggers a CHEK2-p53 DNA damage response, as concomitant deletion of the Trp53 tumor suppressor or Chek2 DNA damage checkpoint kinase rescued Smc5 cKO neurodevelopmental defects. Further assessment using Smc5 cKO and auxin-inducible degron systems demonstrated that absence of SMC5/6 leads to DNA replication stress at late-replicating regions such as pericentromeric heterochromatin. In summary, SMC5/6 is important for completion of DNA replication prior to entering mitosis, which ensures accurate chromosome segregation. Thus, SMC5/6 functions are critical in highly proliferative stem cells during organism development.

Adaptation of the AID system for stem cell and transgenic mouse research.

The auxin-inducible degron (AID) system is becoming a widely used method for rapid and reversible degradation of target proteins. This system has been successfully used to study gene and protein functions in eukaryotic cells and common model organisms, such as nematode and fruit fly. To date, applications of the AID system in mammalian stem cell research are limited. Furthermore, standard mouse models harboring the AID system have not been established. Here we have explored the utility of the H11 safe-harbor locus for integration of the TIR1 transgene, an essential component of auxin-based protein degradation system. We have shown that the H11 locus can support constitutive and conditional TIR1 expression in mouse and human embryonic stem cells, as well as in mice. We demonstrate that the AID system can be successfully employed for rapid degradation of stable proteins in embryonic stem cells, which is crucial for investigation of protein functions in quickly changing environments, such as stem cell proliferation and differentiation. As embryonic stem cells possess unlimited proliferative capacity, differentiation potential, and can mimic organ development, we believe that these research tools will be an applicable resource to a broad scientific audience.

Impact of the Affordable Care Act on Colorectal Cancer Outcomes: A Systematic Review.

CONTEXT: The Patient Protection and Affordable Care Act increases healthcare access and includes provisions that directly impact access to and cost of evidence-based colorectal cancer screening. The Affordable Care Act's removal of cost sharing for colorectal cancer screening as well as Medicaid expansion have been hypothesized to increase screening and improve other health outcomes. However, since its passage in 2010, there is little consensus on the Affordable Care Act's impact. EVIDENCE ACQUISITION: Data from March 2010 to June 2019 were reviewed and 21 relevant studies were identified; 19 studies examined colorectal cancer screening with most finding increased screening rates. EVIDENCE SYNTHESIS: Eleven studies found significant increases, 5 found nonsignificant increases, 3 found nonsignificant decreases, and 1 study found a significant decrease in colorectal cancer screening. Three studies examined the impact on colorectal cancer incidence and stage of diagnosis, where a significant 2.4% increase in early diagnosis was found in one and a nonsignificant increase in incidence in another. However, survival improved after Medicaid expansion. CONCLUSIONS: Free preventive colorectal cancer screening and Medicaid expansion because of passage of the Affordable Care Act have been, in general, positively associated with modest improvements in screening rates across the country. Future studies are needed that investigate the longer-term impact of the Affordable Care Act on colorectal cancer morbidity and mortality rates, as screening is only the first step in treatment of cancerous and precancerous lesions, preventing them from progressing. Moreover, more studies examining subpopulations are needed to better assess where gaps in care remain.

Pharmacological activation of IKr in models of long QT Type 2 risks overcorrection of repolarization.

AIMS: Current treatment for congenital long QT syndrome Type 2 (cLQTS2), an electrical disorder that increases the risk of life-threatening cardiac arrhythmias, is aimed at reducing the incidence of arrhythmia triggers (beta-blockers) or terminating the arrhythmia after onset (implantable cardioverter-defibrillator). An alternative strategy is to target the underlying disease mechanism, which is reduced rapid delayed rectifier current (IKr) passed by Kv11.1 channels. Small molecule activators of Kv11.1 have been identified but the extent to which these can restore normal cardiac signalling in cLQTS2 backgrounds remains unclear. Here, we examined the ability of ICA-105574, an activator of Kv11.1 that impairs transition to the inactivated state, to restore function to heterozygous Kv11.1 channels containing either inactivation enhanced (T618S, N633S) or expression deficient (A422T) mutations. METHODS AND RESULTS: ICA-105574 effectively restored Kv11.1 current from heterozygous inactivation enhanced or expression defective mutant channels in heterologous expression systems. In a human-induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) model of cLQTS2 containing the expression defective Kv11.1 mutant A422T, cardiac repolarization, estimated from the duration of calcium transients in isolated cells and the rate corrected field potential duration (FPDc) in culture monolayers of cells, was significantly prolonged. The Kv11.1 activator ICA-105574 was able to reverse the prolonged repolarization in a concentration-dependent manner. However, at higher doses, ICA-105574 produced a shortening of the FPDc compared to controls. In vitro and in silico analysis suggests that this overcorrection occurs as a result of a temporal redistribution of the peak IKr to much earlier in the plateau phase of the action potential, which results in early repolarization. CONCLUSION: Kv11.1 activators, which target the primary disease mechanism, provide a possible treatment option for cLQTS2, with the caveat that there may be a risk of overcorrection that could itself be pro-arrhythmic.

Dorsal BNST α2A-Adrenergic Receptors Produce HCN-Dependent Excitatory Actions That Initiate Anxiogenic Behaviors.

Stress is a precipitating agent in neuropsychiatric disease and initiates relapse to drug-seeking behavior in addicted patients. Targeting the stress system in protracted abstinence from drugs of abuse with anxiolytics may be an effective treatment modality for substance use disorders. α2A-adrenergic receptors (α2A-ARs) in extended amygdala structures play key roles in dampening stress responses. Contrary to early thinking, α2A-ARs are expressed at non-noradrenergic sites in the brain. These non-noradrenergic α2A-ARs play important roles in stress responses, but their cellular mechanisms of action are unclear. In humans, the α2A-AR agonist guanfacine reduces overall craving and uncouples craving from stress, yet minimally affects relapse, potentially due to competing actions in the brain. Here, we show that heteroceptor α2A-ARs postsynaptically enhance dorsal bed nucleus of the stria terminalis (dBNST) neuronal activity in mice of both sexes. This effect is mediated by hyperpolarization-activated cyclic nucleotide-gated cation channels because inhibition of these channels is necessary and sufficient for excitatory actions. Finally, this excitatory action is mimicked by clozapine-N-oxide activation of the Gi-coupled DREADD hM4Di in dBNST neurons and its activation elicits anxiety-like behavior in the elevated plus maze. Together, these data provide a framework for elucidating cell-specific actions of GPCR signaling and provide a potential mechanism whereby competing anxiogenic and anxiolytic actions of guanfacine may affect its clinical utility in the treatment of addiction.SIGNIFICANCE STATEMENT Stress affects the development of neuropsychiatric disorders including anxiety and addiction. Guanfacine is an α2A-adrenergic receptor (α2A-AR) agonist with actions in the bed nucleus of the stria terminalis (BNST) that produces antidepressant actions and uncouples stress from reward-related behaviors. Here, we show that guanfacine increases dorsal BNST neuronal activity through actions at postsynaptic α2A-ARs via a mechanism that involves hyperpolarization-activated cyclic nucleotide gated cation channels. This action is mimicked by activation of the designer receptor hM4Di expressed in the BNST, which also induces anxiety-like behaviors. Together, these data suggest that postsynaptic α2A-ARs in BNST have excitatory actions on BNST neurons and that these actions can be phenocopied by the so-called "inhibitory" DREADDs, suggesting that care must be taken regarding interpretation of data obtained with these tools.

Perianal Lesions in Children: An Updated Review.

Perianal lesions in children are common reasons for dermatology clinic visits and a well-defined approach to diagnosis and management is helpful to the practicing clinician. In this article, we review and update various etiologies of perianal lesions in the pediatric population, including infectious, papulosquamous, vascular, and neoplastic. We provide a standard initial approach to diagnosis and updates on current management. Infectious etiologies of perianal lesions discussed in this article include fungal, bacterial, parasitic, and viral. Perianal papulosquamous lesions often encountered in children, and discussed in this article, include acrodermatitis enteropathica, psoriasis, contact dermatitis, and many others. We also discuss the diagnosis and management of other entities including infantile hemangiomas, Langerhans cell histiocytosis, and fibrous hamartoma of infancy.

Incontinentia Pigmenti presenting as a newborn eruption: two case presentations.

Linear vesicles or papules in a newborn can be a presenting sign of incontinentia pigmenti (IP). In this report, we present two cases of neonates with cutaneous manifestations of incontinentia pigmenti. In one case, mild peripheral eosinophilia was noted. No extra-cutaneous manifestations were noted otherwise in both cases after complete ophthalmological and neurological evaluations. These cases serve as a reminder for clinicians to consider IP in newborns presenting with linear vesicles or papules.

Unilateral indurated plaque in the axilla: a case of metastatic breast carcinoma.

Breast cancer is the most commonly diagnosed cancer among American women and is also the most common internal malignancy to metastasize to the skin. Rarely, cutaneous metastases represent the first indication of breast carcinoma, putting dermatologists in an instrumental position to make the diagnosis of breast carcinoma. We report the case of a 71-year-old woman with a 10-year history of a slowly-enlarging, indurated plaque in the right axilla. Review of symptoms was significant only for occasional numbness and tingling that extended from the right axilla to the right hand. Biopsy revealed cells infiltrating in a single-file between the collagen bundles in the dermis and subcutis and immunohistochemical staining consistent with a diagnosis of invasive lobular carcinoma. Subsequent work up revealed a primary breast lesion and extensive bony metastases.

Assessing the reliability and validity of direct observation and traffic camera streams to measure helmet and motorcycle use.

There is a need to develop motorcycle helmet surveillance approaches that are less labour intensive than direct observation (DO), which is the commonly recommended but never formally validated approach, particularly in developing settings. This study sought to assess public traffic camera feeds as an alternative to DO, in addition to the reliability of DO under field conditions. DO had high inter-rater reliability, κ=0.88 and 0.84, respectively, for cycle type and helmet type, which reinforces its use as a gold standard. However, traffic camera-based data collection was found to be unreliable, with κ=0.46 and 0.53 for cycle type and helmet type. When bicycles, motorcycles and scooters were classified based on traffic camera streams, only 68.4% of classifications concurred with those made via DO. Given the current technology, helmet surveillance via traffic camera streams is infeasible, and there remains a need for innovative traffic safety surveillance approaches in low-income urban settings.

Copy number variation analysis based on AluScan sequences.

BACKGROUND: AluScan combines inter-Alu PCR using multiple Alu-based primers with opposite orientations and next-generation sequencing to capture a huge number of Alu-proximal genomic sequences for investigation. Its requirement of only sub-microgram quantities of DNA facilitates the examination of large numbers of samples. However, the special features of AluScan data rendered difficult the calling of copy number variation (CNV) directly using the calling algorithms designed for whole genome sequencing (WGS) or exome sequencing. RESULTS: In this study, an AluScanCNV package has been assembled for efficient CNV calling from AluScan sequencing data employing a Geary-Hinkley transformation (GHT) of read-depth ratios between either paired test-control samples, or between test samples and a reference template constructed from reference samples, to call the localized CNVs, followed by use of a GISTIC-like algorithm to identify recurrent CNVs and circular binary segmentation (CBS) to reveal large extended CNVs. To evaluate the utility of CNVs called from AluScan data, the AluScans from 23 non-cancer and 38 cancer genomes were analyzed in this study. The glioma samples analyzed yielded the familiar extended copy-number losses on chromosomes 1p and 9. Also, the recurrent somatic CNVs identified from liver cancer samples were similar to those reported for liver cancer WGS with respect to a striking enrichment of copy-number gains in chromosomes 1q and 8q. When localized or recurrent CNV-features capable of distinguishing between liver and non-liver cancer samples were selected by correlation-based machine learning, a highly accurate separation of the liver and non-liver cancer classes was attained. CONCLUSIONS: The results obtained from non-cancer and cancerous tissues indicated that the AluScanCNV package can be employed to call localized, recurrent and extended CNVs from AluScan sequences. Moreover, both the localized and recurrent CNVs identified by this method could be subjected to machine-learning selection to yield distinguishing CNV-features that were capable of separating between liver cancers and other types of cancers. Since the method is applicable to any human DNA sample with or without the availability of a paired control, it can also be employed to analyze the constitutional CNVs of individuals.

Determination of renal hypothermic temperature adequacy for renoprotection during ischemia using renal interstitial glycerol concentrations in a porcine model.

OBJECTIVES: To determine the effect of renal cooling on interstitial glycerol concentration during renal ischemia. The rate of cellular release of glycerol into the interstitial fluid at various hypothermic temperatures during ischemia was used to assess adequacy for renoprotection at those temperatures. METHODS: Twenty-four renal units in 12 pigs underwent ischemia during measurement of renal interstitial fluid glycerol concentration. Kidneys were categorized into a body temperature control group or various hypothermic temperature groups (n = 4): 5°, 10°, 15°, 20°, and 25°. RESULTS: The glycerol concentration of all kidneys increased directly with ischemic time. The rate of increase in glycerol concentrations over ischemic time decreased sequentially as renal temperature decreased. The glycerol concentration of the kidneys cooled to 25°C during ischemia was significantly less (P = .03) relative to the glycerol levels obtained from the kidneys subjected to warm ischemia at 120 minutes. CONCLUSIONS: Renal hypothermia decreases the rate of cellular release of glycerol into the interstitial fluid. Hypothermia at 25°C doubles the time required for renal interstitial glycerol to accumulate to levels associated with irreparable renal function damage. Therefore, relatively warmer hypothermic temperatures may be sufficient to extend a significant renoprotective effect during ischemia.

Targeting YAP and Hippo signaling pathway in liver cancer.

IMPORTANCE OF THE FIELD: The Hippo signaling pathway plays pivotal roles in controlling both cell growth and organ size, emerging as a new paradigm in tumor suppression. Yes-associated protein (YAP) functions as a potent transcription co-activator and is a major downstream target tightly regulated by the Hippo pathway. Inactivation of the Hippo signaling induces YAP-mediated activation of various target genes that functionally causes cellular proliferation and outgrowth of organ size. Recently, YAP has been implicated as a bona fide oncogene in solid tumors, but little is known about its exact molecular mechanism in carcinogenesis. AREAS COVERED IN THIS REVIEW: We discuss the latest important findings in the Hippo signaling pathway and the possible means of developing potential cancer therapeutics by targeting multiple sites along the Hippo pathway. WHAT THE READER WILL GAIN: An overview of the emerging roles of YAP and Hippo signaling in oncogenesis and the possible ways of developing cancer therapies against the pathway components, downstream targets or interconnected pathways. TAKE HOME MESSAGE: YAP is a key oncogenic driver in liver carcinogenesis and deregulation of the Hippo pathway causes tumor formation and malignancy. Targeting YAP and cognate downstream signaling targets may have clinical utility in cancer therapies.

Pillar-array based optical sensor.

An optical microcavity based on pillar arrays has been fabricated in Si/SiO(2) material system. Transmission measurement was taken and a quality factor as high as 27,600 was observed. This cavity was tested for sensing applications by immersing into optical fluids with accurate refractive indices. For refractive index change of 0.01, a resonance peak wavelength shift of 3.5 nm was measured. We also compare cavities consisting of pillars with different aspect ratios.

Circulating Lamin B1 (LMNB1) biomarker detects early stages of liver cancer in patients.

Hepatocellular carcinoma (HCC) is a major liver malignancy possessing a high mortality rate and is particularly prevalent in China and Asia. While surgery is the most effective treatment for liver tumor, about 80% of HCC patients are inoperable at presentation and die early due to late diagnosis. For early cancer detection, we employed a proteomic expression profiling approach to identify biomarkers for early stages of HCC and subsequently assessed the clinical feasibility of a novel marker in plasma. Frozen liver tissues from a retrospective cohort of 75 liver patients (39 HCCs, 20 cirrhosis, and 16 nondiseased subjects) were subjected to proteome-wide expression profiling by 2-DE. MALDI-TOF/TOF was used to identify differentially expressed proteins, which were further confirmed by immunoblotting, qPCR, and immunohistochemistry. Conventional RT-PCR was employed to further analyze the abundance of selected biomarker at mRNA level in a separate cohort of 63 plasma samples (35 HCCs, 16 liver cirrhosis, 12 healthy individuals). We successfully identified lamin B1 (LMNB1) that was significantly upregulated in HCC tumors and present in patients' plasma. LMNB1 functions in nuclear envelope lamina and possesses a transcriptional coregulatory activity having an important role in DNA replication, cellular aging, and stress responses. Clinically, the expression level of lamin B1 correlated positively with tumor stages, tumor sizes, and number of nodules. Our findings further showed elevation of circulating LMNB1 marker in plasma could detect early stages of HCC patients, with 76% sensitivity and 82% specificity. In conclusion, lamin B1 is a clinically useful biomarker for early stages of HCC in tumor tissues and plasma, and warrants further clinical investigation.