HDAC activity is dispensable for repression of cell-cycle genes by DREAM and E2F:RB complexes.

Histone deacetylases (HDACs) play a crucial role in transcriptional regulation and are implicated in various diseases, including cancer. They are involved in histone tail deacetylation and canonically linked to transcriptional repression. Previous studies suggested that HDAC recruitment to cell-cycle gene promoters via the retinoblastoma (RB) protein or the DREAM complex through SIN3B is essential for G1/S and G2/M gene repression during cell-cycle arrest and exit. Here we investigate the interplay among DREAM, RB, SIN3 proteins, and HDACs in the context of cell-cycle gene repression. Knockout of SIN3B does not globally derepress cell-cycle genes in non-proliferating HCT116 and C2C12 cells. Loss of SIN3A/B moderately upregulates several cell-cycle genes in HCT116 cells but does so independently of DREAM/RB. HDAC inhibition does not induce general upregulation of RB/DREAM target genes in arrested transformed or non-transformed cells. Our findings suggest that E2F:RB and DREAM complexes can repress cell-cycle genes without relying on HDAC activity.

Mapping Differential Protein-Protein Interaction Networks using Affinity Purification Mass Spectrometry.

Proteins congregate into complexes to perform fundamental cellular functions. Phenotypic outcomes, in health and disease, are often mechanistically driven by the remodeling of protein complexes by protein-coding mutations or cellular signaling changes in response to molecular cues. Here, we present an affinity purification-mass spectrometry (APMS) proteomics protocol to quantify and visualize global changes in protein-protein interaction (PPI) networks between pairwise conditions. We describe steps for expressing affinity-tagged "bait" proteins in mammalian cells, identifying purified protein complexes, quantifying differential PPIs, and visualizing differential PPI networks. Specifically, this protocol details steps for designing affinity-tagged "bait" gene constructs, transfection, affinity purification, mass spectrometry sample preparation, data acquisition, database search, data quality control, PPI confidence scoring, cross-run normalization, statistical data analysis, and differential PPI visualization. Our protocol discusses caveats and limitations with applicability across cell types and biological areas. For complete details on the use and execution of this protocol, please refer to Bouhaddou et al. 20231.

Cleavage-resistant RIPK1-induced autoinflammatory syndrome-A report of three generations with periodic fever and clinical response to colchicine.

The clinical syndrome caused by cleavage-resistant RIPK1 is known as CRIA (Cleavage-resistant RIPK1-induced autoinflammatory) syndrome. We present a family with three generations affected by CRIA syndrome. Our index patient (P1), a boy born of a non-consanguineous marriage, developed recurrent episodes of fever after 5 months of age, with variable periodicity. His father (P2) and paternal grandmother also had periodic fever. At 23 months of age, P1 was diagnosed with renal biopsy-proven steroid-responsive nephrotic syndrome. His first visit to our center was at 2 years of age. At presentation, he had failure to thrive, microcytic hypochromic anemia, and elevated inflammatory markers and interleukin-6 levels. Amyloid A protein was elevated, serum creatinine was normal, and proteinuria resolved after addition of steroids. Next-generation sequencing showed heterozygous mutation (c.970G>A, p.Asp324His) in RIPK1. This mutation has been reported to cause CRIA syndrome. P2 and P1's asymptomatic younger brother had the same mutation. All the affected members showed variability with respect to frequency and duration of periodic fever as well as the age of onset. Both P1 and P2 had elevated amyloid A, with no evidence of renal dysfunction. P1 and P2 showed improvement in the intensity of fever spikes with colchicine treatment; however, both continue to have periodic fever.

HDAC activity is dispensable for repression of cell-cycle genes by DREAM and E2F:RB complexes.

Histone deacetylases (HDACs) are pivotal in transcriptional regulation, and their dysregulation has been associated with various diseases including cancer. One of the critical roles of HDAC-containing complexes is the deacetylation of histone tails, which is canonically linked to transcriptional repression. Previous research has indicated that HDACs are recruited to cell-cycle gene promoters through the RB protein or the DREAM complex via SIN3B and that HDAC activity is essential for repressing G1/S and G2/M cell-cycle genes during cell-cycle arrest and exit. In this study, we sought to explore the interdependence of DREAM, RB, SIN3 proteins, and HDACs in the context of cell-cycle gene repression. We found that genetic knockout of SIN3B did not lead to derepression of cell-cycle genes in non-proliferating HCT116 and C2C12 cells. A combined loss of SIN3A and SIN3B resulted in a moderate upregulation in mRNA expression of several cell-cycle genes in arrested HCT116 cells, however, these effects appeared to be independent of DREAM or RB. Furthermore, HDAC inhibition did not induce a general upregulation of RB and DREAM target gene expression in arrested transformed or non-transformed cells. Our findings provide evidence that E2F:RB and DREAM complexes can repress cell-cycle genes without reliance on HDAC activity.

SARS-CoV-2 variants evolve convergent strategies to remodel the host response.

SARS-CoV-2 variants of concern (VOCs) emerged during the COVID-19 pandemic. Here, we used unbiased systems approaches to study the host-selective forces driving VOC evolution. We discovered that VOCs evolved convergent strategies to remodel the host by modulating viral RNA and protein levels, altering viral and host protein phosphorylation, and rewiring virus-host protein-protein interactions. Integrative computational analyses revealed that although Alpha, Beta, Gamma, and Delta ultimately converged to suppress interferon-stimulated genes (ISGs), Omicron BA.1 did not. ISG suppression correlated with the expression of viral innate immune antagonist proteins, including Orf6, N, and Orf9b, which we mapped to specific mutations. Later Omicron subvariants BA.4 and BA.5 more potently suppressed innate immunity than early subvariant BA.1, which correlated with Orf6 levels, although muted in BA.4 by a mutation that disrupts the Orf6-nuclear pore interaction. Our findings suggest that SARS-CoV-2 convergent evolution overcame human adaptive and innate immune barriers, laying the groundwork to tackle future pandemics.

Impact of SARS-CoV-2 ORF6 and its variant polymorphisms on host responses and viral pathogenesis.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) encodes several proteins that inhibit host interferon responses. Among these, ORF6 antagonizes interferon signaling by disrupting nucleocytoplasmic trafficking through interactions with the nuclear pore complex components Nup98-Rae1. However, the roles and contributions of ORF6 during physiological infection remain unexplored. We assessed the role of ORF6 during infection using recombinant viruses carrying a deletion or loss-of-function (LoF) mutation in ORF6. ORF6 plays key roles in interferon antagonism and viral pathogenesis by interfering with nuclear import and specifically the translocation of IRF and STAT transcription factors. Additionally, ORF6 inhibits cellular mRNA export, resulting in the remodeling of the host cell proteome, and regulates viral protein expression. Interestingly, the ORF6:D61L mutation that emerged in the Omicron BA.2 and BA.4 variants exhibits reduced interactions with Nup98-Rae1 and consequently impairs immune evasion. Our findings highlight the role of ORF6 in antagonizing innate immunity and emphasize the importance of studying the immune evasion strategies of SARS-CoV-2.

A Kinematic Analysis of Wrist and Carpal Function Using Four-Dimensional Computed Tomography Technology: A Dynamic Perspective.

PURPOSE: An emerging imaging modality, four-dimensional computed tomography, can provide dynamic evaluation of carpal motion, which allows for a better understanding of how the carpals work together to achieve range of motion. The objective of this work was to examine kinematic motion of the carpus through a flexion/extension arc of motion using four-dimensional computed tomography. METHODS: A convenience sample of 20 uninjured participants underwent a four-dimensional computed tomography scanning protocol through a complete arc of flexion/extension motion. Kinematic changes in motion were quantified using helical axes motion data for each carpal. Rotation angles were compared between bones to identify differences in kinematic motion between bones. RESULTS: The bones within the proximal carpal row, the lunate, scaphoid, and triquetrum, rotate significantly to differing magnitudes at the ends of motion (40° of flexion and 40° of extension). The scaphoid rotates to the highest magnitude, followed by the triquetrum, and lastly, the lunate. The distal carpal row bones rotate to similar magnitudes throughout the entire range of motion. CONCLUSIONS: This work describes the kinematics of the carpals throughout dynamic in vivo flexion and extension. CLINICAL RELEVANCE: This study adds to an understanding of wrist mechanics and the possible clinical implications of pathological deviation from baseline kinematics.

The Impact of Periodic Formative Assessments on Learning Through the Lens of the Complex Adaptive System and Social Sustainability Principles.

PURPOSE: Medical institutions in India have employed different periodic formative assessment (FA) methods with variable impact. The formative evaluation must incorporate feedback to improve learning. Formative assessment has helped inexperienced students apprehend their weaknesses, make choices, prepare for summative exams, and allow teachers to identify regions wherein students may also need aid. This study attempts to quantify the impact of the weekly, monthly, and semester formative assessments and view it retrospectively through the lens of complex adaptive systems and social sustainability principles. METHODS: We used a post-exam survey and statistical analysis to compare the students' performance between the timely periodic formative assessments in a competency-based curriculum. The cohort consisted of 2018 (semester), 2019 (weekly), and 2020 (monthly) first-year medical students. Cronbach alpha, spearman's correlation coefficient, descriptive statistics, and repeated measure analysis of variance were used to explore the reliability and relationship between formative assessment and summative scores of each cohort and find any significant difference. The authors also analyzed the accordance between the FA exam performance and students' perceptions, deduced broad themes, and discussed the appropriateness and feasibility of students' suggestions for changes. RESULTS: A significant correlation was found only between the weekly formative assessment and summative scores (r=0.74, p=0.01). The analysis of variance established significant differences between all summative scores of respective periodic formative assessments. The weekly formative assessment showed the highest mean summative examination scores. This study helped comprehend preclinical students' apprehension after the intervention of periodic formative assessments. The students found this intervention helpful in driving and detecting gaps in learning but preferred focused feedback, clinically oriented practices, and countered mental health issues. CONCLUSION: The continuous periodic formative assessment model had a valid educational impact but was not sustainable according to social sustainability principles. A complex adaptive framework can be utilized to make it sustainable.

Clinical, immunological and molecular findings of patients with DOCK-8 deficiency from India.

DOCK8 deficiency affects various cell subsets belonging to both the innate and adaptive immune systems. Clinical diagnosis is challenging, as many cases present with severe atopic dermatitis as the only initial manifestation. Though flow cytometry helps in the presumptive diagnosis of DOCK8-deficient patients by evaluating their DOCK8 protein expression, it requires subsequent confirmation by molecular genetic analysis. Currently, haematopoietic stem cell transplantation (HSCT) is the only curative treatment option available for these patients. There is a paucity of data from India on the clinical diversity and molecular spectrum of DOCK8 deficiency. In the present study, we report the clinical, immunological and molecular findings of 17 DOCK8-deficient patients from India diagnosed over the last 5 years.

Inadequate proximal screw fixation increases risk of failure following plate fixation of diaphyseal humerus fractures.

BACKGROUND: Operative treatment of humeral shaft fractures (AO/OTA 12) is being performed more frequently. Accordingly, it is important to understand the complications associated with plate fixation. This study analyzes risk factors associated with mechanical failure following plate fixation of humeral shaft fractures in order to further elucidate the mode and location of failure. METHODS: A retrospective review of 351 humeral shaft fractures was completed at a single level I trauma center. Eleven of eighty-five humeral shaft fractures had aseptic mechanical failure requiring revision (12.9%), following initial plate fixation. Fracture characteristics (AO type, comminution, location) and fracture fixation (plate type, multiplanar, number of screws proximal and distal to the fracture) were compared between aseptic mechanical failure and those without failure. A forward stepwise logistic regression analysis was performed to determine any significant predictors of aseptic mechanical failure. RESULTS: There was significant differences in fixation between the aseptic mechanical failure group and those without failure, specifically in the number of screws for proximal fixation (p = 0.008) and distal fixation (p = 0.040). In the aseptic mechanical failure group, patients tended to have less than < 8 cortices of proximal fixation (82%) and less than < 8 cortices of distal fixation (64%). Conversely, in patients without mechanical failure there was a tendency to have greater than > 8 cortices in both the proximal (62%) and distal fixation (70%). A forward stepwise logistic regression analysis found that less than < 8 cortices of proximal fixation was a significant predictor of aseptic failure, OR 7.96 (p = 0.011). We think this can be accounted for due to the variable bone quality, thinner cortices and multiple torsional forces in the proximal shaft that may warrant special consideration for fixation. CONCLUSION: The current dogma of humeral shaft fracture stabilization is to use a minimum of 3 screws proximal and distal to the fracture, however the current study demonstrates this is associated with higher rates of mechanical failure. In contrast, 4 bicortical screws or more of fixation on either side of the fracture had lower failure rates and may help to reduce the risk of mechanical failure. Level of Evidence Level III.

Accessory Lobe of Liver: A Rare Hepatic Variation With Diagnostic Dilemma.

The liver is a very dynamic organ. Still, the gross anomalies of the liver are sparse. The accessory lobe of the liver is a rare anatomical variation with a prevalence of less than 1%. We present a case of an accessory lobe and two accessory grooves of the liver in a cadaver. The accessory lobe was an isolated anomaly, sessile in presentation, and attached to normal liver parenchyma. Although accessory lobes are rare, knowledge about them will reiterate to surgeons and radiologists to be mindful of them and to avoid misdiagnosis.

A comprehensive study of p53 protein.

The protein p53 has been extensively investigated since it was found 43 years ago and has become a "guardian of the genome" that regulates the division of cells by preventing the growth of cells and dividing them, that is, inhibits the development of tumors. Initial proof of protein existence by researchers in the mid-1970s was found by altering and regulating the SV40 big T antigen termed the A protein. Researchers demonstrated how viruses play a role in cancer by employing viruses' ability to create T-antigens complex with viral tumors, which was discovered in 1979 following a viral analysis and cancer analog research. Researchers later in the year 1989 explained that in Murine Friend, a virus-caused erythroleukemia, commonly found that p53 was inactivated to suggest that p53 could be a "tumor suppressor gene." The TP53 gene, encoding p53, is one of human cancer's most frequently altered genes. The protein-regulated biological functions of all p53s include cell cycles, apoptosis, senescence, metabolism of the DNA, angiogenesis, cell differentiation, and immunological response. We tried to unfold the history of the p53 protein, which was discovered long back in 1979, that is, 43 years of research on p53, and how p53's function has been developed through time in this article.

Impact of SARS-CoV-2 ORF6 and its variant polymorphisms on host responses and viral pathogenesis.

We and others have previously shown that the SARS-CoV-2 accessory protein ORF6 is a powerful antagonist of the interferon (IFN) signaling pathway by directly interacting with Nup98-Rae1 at the nuclear pore complex (NPC) and disrupting bidirectional nucleo-cytoplasmic trafficking. In this study, we further assessed the role of ORF6 during infection using recombinant SARS-CoV-2 viruses carrying either a deletion or a well characterized M58R loss-of-function mutation in ORF6. We show that ORF6 plays a key role in the antagonism of IFN signaling and in viral pathogenesis by interfering with karyopherin(importin)-mediated nuclear import during SARS-CoV-2 infection both in vitro , and in the Syrian golden hamster model in vivo . In addition, we found that ORF6-Nup98 interaction also contributes to inhibition of cellular mRNA export during SARS-CoV-2 infection. As a result, ORF6 expression significantly remodels the host cell proteome upon infection. Importantly, we also unravel a previously unrecognized function of ORF6 in the modulation of viral protein expression, which is independent of its function at the nuclear pore. Lastly, we characterized the ORF6 D61L mutation that recently emerged in Omicron BA.2 and BA.4 and demonstrated that it is able to disrupt ORF6 protein functions at the NPC and to impair SARS-CoV-2 innate immune evasion strategies. Importantly, the now more abundant Omicron BA.5 lacks this loss-of-function polymorphism in ORF6. Altogether, our findings not only further highlight the key role of ORF6 in the antagonism of the antiviral innate immune response, but also emphasize the importance of studying the role of non-spike mutations to better understand the mechanisms governing differential pathogenicity and immune evasion strategies of SARS-CoV-2 and its evolving variants. ONE SENTENCE SUMMARY: SARS-CoV-2 ORF6 subverts bidirectional nucleo-cytoplasmic trafficking to inhibit host gene expression and contribute to viral pathogenesis.

Global landscape of the host response to SARS-CoV-2 variants reveals viral evolutionary trajectories

A series of SARS-CoV-2 variants of concern (VOCs) have evolved in humans during the COVID-19 pandemic--Alpha, Beta, Gamma, Delta, and Omicron. Here, we used global proteomic and genomic analyses during infection to understand the molecular responses driving VOC evolution. We discovered VOC-specific differences in viral RNA and protein expression levels, including for N, Orf6, and Orf9b, and pinpointed several viral mutations responsible. An analysis of the host response to VOC infection and comprehensive interrogation of altered virus-host protein-protein interactions revealed conserved and divergent regulation of biological pathways. For example, regulation of host translation was highly conserved, consistent with suppression of VOC replication in mice using the translation inhibitor plitidepsin. Conversely, modulation of the host inflammatory response was most divergent, where we found Alpha and Beta, but not Omicron BA.1, antagonized interferon stimulated genes (ISGs), a phenotype that correlated with differing levels of Orf6. Additionally, Delta more strongly upregulated proinflammatory genes compared to other VOCs. Systematic comparison of Omicron subvariants revealed BA.5 to have evolved enhanced ISG and proinflammatory gene suppression that similarly correlated with Orf6 expression, effects not seen in BA.4 due to a mutation that disrupts the Orf6-nuclear pore interaction. Our findings describe how VOCs have evolved to fine-tune viral protein expression and protein-protein interactions to evade both innate and adaptive immune responses, offering a likely explanation for increased transmission in humans. One sentence summarySystematic proteomic and genomic analyses of SARS-CoV-2 variants of concern reveal how variant-specific mutations alter viral gene expression, virus-host protein complexes, and the host response to infection with applications to therapy and future pandemic preparedness.

Impact of SARS-CoV-2 ORF6 and its variant polymorphisms on host responses and viral pathogenesis.

We and others have previously shown that the SARS-CoV-2 accessory protein ORF6 is a powerful antagonist of the interferon (IFN) signaling pathway by directly interacting with Nup98-Rae1 at the nuclear pore complex (NPC) and disrupting bidirectional nucleo-cytoplasmic trafficking. In this study, we further assessed the role of ORF6 during infection using recombinant SARS-CoV-2 viruses carrying either a deletion or a well characterized M58R loss-of-function mutation in ORF6. We show that ORF6 plays a key role in the antagonism of IFN signaling and in viral pathogenesis by interfering with karyopherin(importin)-mediated nuclear import during SARS-CoV-2 infection both in vitro, and in the Syrian golden hamster model in vivo. In addition, we found that ORF6-Nup98 interaction also contributes to inhibition of cellular mRNA export during SARS-CoV-2 infection. As a result, ORF6 expression significantly remodels the host cell proteome upon infection. Importantly, we also unravel a previously unrecognized function of ORF6 in the modulation of viral protein expression, which is independent of its function at the nuclear pore. Lastly, we characterized the ORF6 D61L mutation that recently emerged in Omicron BA.2 and BA.4 and demonstrated that it is able to disrupt ORF6 protein functions at the NPC and to impair SARS-CoV-2 innate immune evasion strategies. Importantly, the now more abundant Omicron BA.5 lacks this loss-of-function polymorphism in ORF6. Altogether, our findings not only further highlight the key role of ORF6 in the antagonism of the antiviral innate immune response, but also emphasize the importance of studying the role of non-spike mutations to better understand the mechanisms governing differential pathogenicity and immune evasion strategies of SARS-CoV-2 and its evolving variants. ONE SENTENCE SUMMARYSARS-CoV-2 ORF6 subverts bidirectional nucleo-cytoplasmic trafficking to inhibit host gene expression and contribute to viral pathogenesis.

Role of Oxidative Stress and the Protective Effect of Fermented Papaya Preparation in Sickle Cell Disease.

Fermented papaya preparation (FPP) is the source of antioxidants that may help in reducing the complications associated with oxidative stress and may improve the quality of life in sickle cell disease patients. In this study, we assessed the in vitro effect of FPP on sickled red blood cells (RBCs) using oxidative stress markers and observed that FPP has the potential to reduce the oxidative stress. Scanning electron microscopy (SEM) and eosin 5' malaemide (E5'M) dye test showed that FPP protects red cell morphology against the oxidative stress. Liquid chromatography mass spectrometry (LCMS) analysis of FPP suggests the presence of essential amino acids, vitamin D3, and its derivatives. Fermented papaya preparation can be of benefit either in reducing oxidative stress parameters or in preventing pathophysiological events in the sickle cell disease patients.

Comparative Morphometry of the Sacrum and Its Clinical Implications: A Retrospective Study of Osteometry in Dry Bones and CT Scan Images in Patients Presenting With Lumbosacral Pathologies.

Background Morphometric measurement of the sacrum is crucial due to its active involvement in the instrumentation for lumbar pathologies. From screw placement to stabilization procedures for the spine, the sacrum remains a site of surgical importance. Thus, the purpose of this study was to generate baseline data by comparing two techniques, namely, osteometry in dry bones and CT scan imaging. Methodology In this study, 30 dry, fully ossified, disarticulated sacra were studied for osteometry, and 60 CT scan reports of patients with lumbar pathologies were retrospectively evaluated. In both cases, similar parameters were measured. The mean values were determined, the two methods were compared, and statistical analysis was performed. Results Among the 30 dry bone samples, 33.3% (10 out of 30) were males, while 55% of the CT scan group were males. Correlation between the different measurements in the CT scan group suggested that the vertebral body maximum width of S1 had a significant positive correlation with the vertebral body height of S1, sacral height, sacral breadth, transverse diameter of auricular surface, and vertical diameter of auricular surface. Statistically significant higher values (P < 0.001) were observed for the vertebral body mid diameter of S1, vertebral body height of S1, pedicle width, and pedicle depth measurements in the dry bone group compared to the CT scan group. Conclusions The efficiency of anaesthetic blocks can be increased if the parameters are evaluated beforehand. Moreover, sexual dimorphism of the bone can account for the varied results of the parameters, indicating the necessity to conduct gender-based studies in a wider population.

Sex estimation from dimensions of the base of the skull in Black South Africans.

The pelvis and the skull are the two most utilised skeletal elements to estimate sex from skeletonised remains due to their sexually dimorphic traits. However, as increasingly more fragmented remains have been presented for analyses, other bones and their fragments have now been subjected to analyses for sex estimation. In the skull particularly, the base has shown to survive harsh conditions. In this study the foramen magnum region was explored in Black South Africans to estimate sex during forensic analyses. Seven measurements of the foramen magnum and surrounding areas were measured in 120 male and female crania and subjected to discriminant function analyses. The average accuracies for the stepwise discriminant functions ranged from 60-71% whilst the average accuracies for the direct discriminant functions ranged from 63-69%. The average accuracies obtained in this study are similar to other studies performed using the foramen magnum. However, these average accuracies are much lower than other skeletal elements that have been used for sex estimation in South Africans. Thus, the equations in this study should be used with caution and only in the absence of more accurate elements. The cranial base has always shown to have a low to moderate expression of sexual dimorphism. The cranial base of Black South Africans is no different.

Transoral endoscopic repair of Zenker diverticulum by a thoracic surgical service.

OBJECTIVE: Zenker diverticulum (ZD), a pulsion diverticulum of the esophagus, has been traditionally managed with an open surgical approach, but endoscopic transoral stapling has been reported with increasing frequency. The objective of this study was to evaluate the results of endoscopic repair of ZD by a thoracic surgery service. METHODS: We conducted a retrospective review of patients who underwent transoral stapling repair of ZD at our institution by the thoracic surgery service. We evaluated perioperative outcomes including dysphagia (1, no dysphagia to 5, unable to swallow saliva) and failure of repair requiring surgical intervention. RESULTS: A total of 151 patients (median age, 78 years; 75 men, 76 women) underwent evaluation for endoscopic repair of ZD. Endoscopic stapled repair of the ZD was completed in 135. Sixteen patients underwent conversion to open repair. The perioperative mortality was 0.6% (1 patient). The median hospital stay was 2 days (range, 0-18 days). Complications occurred in 5 patients who underwent endoscopic repair. The mean preoperative dysphagia score was 2.8 and improved to 1.2 during follow-up (median, 16 months; P < .001). During further follow-up (median, 52 months), 8 patients (5.3%) had failure of the endoscopic repair requiring open surgery (n = 5) or redo transoral stapling (n = 3). CONCLUSIONS: Endoscopic stapling repair of ZD can be performed safely with good results in experienced centers by thoracic surgeons with significant esophageal experience. Long-term follow-up is required to evaluate the durability of endoscopic repair of ZD.