Suppression of ferroptosis by vitamin A or radical-trapping antioxidants is essential for neuronal development.

The development of functional neurons is a complex orchestration of multiple signaling pathways controlling cell proliferation and differentiation. Because the balance of antioxidants is important for neuronal survival and development, we hypothesized that ferroptosis must be suppressed to gain neurons. We find that removal of antioxidants diminishes neuronal development and laminar organization of cortical organoids, which is fully restored when ferroptosis is inhibited by ferrostatin-1 or when neuronal differentiation occurs in the presence of vitamin A. Furthermore, iron-overload-induced developmental growth defects in C. elegans are ameliorated by vitamin E and A. We determine that all-trans retinoic acid activates the Retinoic Acid Receptor, which orchestrates the expression of anti-ferroptotic genes. In contrast, retinal and retinol show radical-trapping antioxidant activity. Together, our study reveals an unexpected function of vitamin A in coordinating the expression of essential cellular gatekeepers of ferroptosis, and demonstrates that suppression of ferroptosis by radical-trapping antioxidants or by vitamin A is required to obtain mature neurons and proper laminar organization in cortical organoids.

A protocol for CRISPR-mediated activation and repression of human endogenous retroviruses in human pluripotent stem cells.

Human endogenous retroviruses (HERVs) comprise many regulatory elements and can regulate host gene activity at different expression levels via multiple mechanisms. Here, we introduce a step-by-step protocol to activate or repress transcription of HERV-K(HML-2) elements using the CRISPRa and CRISPRi technologies in human embryonic stem cells. This protocol can help deciphering the functional role of HERV-K(HML-2) elements in critical biological processes. The protocol may easily be adapted to other cell lines and HERV groups with relatively low sequence heterogeneity. For complete details on the use and execution of this protocol, please refer to Padmanabhan Nair et al. (2021).

Deciphering the association of intronic single nucleotide polymorphisms of crystallin gene family with congenital cataract.

Purpose: Introns play an important role in gene regulation and expression. Single nucleotide polymorphisms (SNPs) in introns have the potential to cause disease and alter the genotype-phenotype association. Hence, this study aimed to decipher the association of SNPs in the introns of the crystallin gene in congenital cataracts. Methods: SNPs in the introns of crystallin gene family - CRYAA (rs3788059), CRYAB (rs2070894), CRYBA4 (rs2071861), and CRYBB2 (rs5752083, rs5996863) - were genotyped in 248 participants consisting of 141 congenital cataracts and 107 healthy controls by allele-specific oligonucleotide polymerase chain reaction method. Around 10% of samples for each SNPs were sequenced to confirm the genotypes. The allele, genotype, and haplotype frequency were evaluated by the SHEsis online tool. Results: Using dominant model, the "A" allele of rs3788059 was found to have an increased risk toward congenital cataract development whereas the "G" allele was found to be protective (AA + AG vs. GG; odds ratio [OR] 95% confidence interval [CI] = 3.73 [1.71, 8.15], P = 0.0009). The "A" allele of both rs2070894 (AA + AG vs. GG; OR [95% CI] = 0.49 [0.29, 0.84], P = 0.012) and rs5752083 (AA + AC vs. CC; OR [95% CI] = 0.25 [0.08, 0.76], P = 0.016) were suggested to have a protective role by the dominant model. The A-C-T haplotype (rs2071861, rs5752083, and rs5996863) was found to be a significant risk factor for the development of congenital cataract. Conclusion: Intronic SNPs in crystallin genes may play a role in the predisposition toward congenital cataract. However, the present findings need to be replicated in a large cohort with more number of samples.

Activation of HERV-K(HML-2) disrupts cortical patterning and neuronal differentiation by increasing NTRK3.

The biological function and disease association of human endogenous retroviruses (HERVs) are largely elusive. HERV-K(HML-2) has been associated with neurotoxicity, but there is no clear understanding of its role or mechanistic basis. We addressed the physiological functions of HERV-K(HML-2) in neuronal differentiation using CRISPR engineering to activate or repress its expression levels in a human-pluripotent-stem-cell-based system. We found that elevated HERV-K(HML-2) transcription is detrimental for the development and function of cortical neurons. These effects are cell-type-specific, as dopaminergic neurons are unaffected. Moreover, high HERV-K(HML-2) transcription alters cortical layer formation in forebrain organoids. HERV-K(HML-2) transcriptional activation leads to hyperactivation of NTRK3 expression and other neurodegeneration-related genes. Direct activation of NTRK3 phenotypically resembles HERV-K(HML-2) induction, and reducing NTRK3 levels in context of HERV-K(HML-2) induction restores cortical neuron differentiation. Hence, these findings unravel a cell-type-specific role for HERV-K(HML-2) in cortical neuron development.

Increased HERV-K(HML-2) Transcript Levels Correlate with Clinical Parameters of Liver Damage in Hepatitis C Patients.

Chronic hepatitis C virus (HCV) infection is closely associated with a plethora of diseases, including cancers and autoimmune disorders. However, the distinct triggers and cellular networks leading to such HCV-derived diseases are poorly understood. Around 8% of the human genome consists of human endogenous retroviruses. They are usually silenced but can be reactivated by environmental conditions, including viral infections. Our current understanding indicates that the activation of one specific family-namely, HERV-K(HML-2)-is linked to distinct pathologies, including cancer and autoimmunity. In this study, we analyzed the transcription levels of HERV-K(HML-2) in 42 HCV-infected patients receiving direct-acting antiviral therapies. Samples from the start of treatment until 12 weeks post-treatment were investigated. Our results show increased HERV-K(HML-2) transcript levels in patients with HCV-derived liver cirrhosis throughout the observation period. Several clinical parameters specifying poor liver function are positively correlated with HERV-K(HML-2) expression. Of note, patients without a sustained viral clearance showed a drastic increase in HERV-K(HML-2) transcript levels. Together, our data suggest that increased HERV-K(HML-2) expression is correlated with reduced liver function as well as therapy success in HCV-infected patients.

Genome-wide investigation identifies a rare copy-number variant burden associated with human spina bifida.

PURPOSE: Next-generation sequencing has implicated some risk variants for human spina bifida (SB), but the genome-wide contribution of structural variation to this complex genetic disorder remains largely unknown. We examined copy-number variant (CNV) participation in the genetic architecture underlying SB risk. METHODS: A high-confidence ensemble approach to genome sequences (GS) was benchmarked and employed for systematic detection of common and rare CNVs in two separate ancestry-matched SB case-control cohorts. RESULTS: SB cases were enriched with exon disruptive rare CNVs, 44% of which were under 10 kb, in both ancestral populations (P = 6.75 × 10-7; P = 7.59 × 10-4). Genes containing these disruptive CNVs fall into molecular pathways, supporting a role for these genes in SB. Our results expand the catalog of variants and genes with potential contribution to genetic and gene-environment interactions that interfere with neurulation, useful for further functional characterization. CONCLUSION: This study underscores the need for genome-wide investigation and extends our previous threshold model of exonic, single-nucleotide variation toward human SB risk to include structural variation. Since GS data afford detection of CNVs with greater resolution than microarray methods, our results have important implications toward a more comprehensive understanding of the genetic risk and mechanisms underlying neural tube defect pathogenesis.

Host-Virus Protein Interaction Network Reveals the Involvement of Multiple Host Processes in the Life Cycle of Hepatitis E Virus.

Comprehensive knowledge of host-pathogen interactions is central to understand the life cycle of a pathogen and devise specific therapeutic strategies. Protein-protein interactions (PPIs) are key mediators of host-pathogen interactions. Hepatitis E virus (HEV) is a major cause of viral hepatitis in humans. Recent reports also demonstrate its extrahepatic manifestations in the brain. Toward understanding the molecular details of HEV life cycle, we screened human liver and fetal brain cDNA libraries to identify the host interaction partners of proteins encoded by genotype 1 HEV and constructed the virus-host PPI network. Analysis of the network indicated a role of HEV proteins in modulating multiple host biological processes such as stress and immune responses, the ubiquitin-proteasome system, energy and iron metabolism, and protein translation. Further investigations revealed the presence of multiple host translation regulatory factors in the viral translation/replication complex. Depletion of host translation factors such as eIF4A2, eIF3A, and RACK1 significantly reduced the viral replication, whereas eIF2AK4 depletion had no effect. These findings highlight the ingenuity of the pathogen in manipulating the host machinery to its own benefit, a clear understanding of which is essential for the identification of strategic targets and development of specific antivirals against HEV. IMPORTANCE Hepatitis E virus (HEV) is a pathogen that is transmitted by the fecal-oral route. Owing to the lack of an efficient laboratory model, the life cycle of the virus is poorly understood. During the course of infection, interactions between the viral and host proteins play essential roles, a clear understanding of which is essential to decode the life cycle of the virus. In this study, we identified the direct host interaction partners of all HEV proteins and generated a PPI network. Our functional analysis of the HEV-human PPI network reveals a role of HEV proteins in modulating multiple host biological processes such as stress and immune responses, the ubiquitin-proteasome system, energy and iron metabolism, and protein translation. Further investigations revealed an essential role of several host factors in HEV replication. Collectively, the results from our study provide a vast resource of PPI data from HEV and its human host and identify the molecular components of the viral translation/replication machinery.

Pulmonary mucormycosis diagnosed by convex probe endobronchial ultrasound-guided fine needle aspiration of cavity wall.

Pulmonary mucormycosis is an opportunistic fungal infection in immunocompromised individuals. It is difficult to diagnose as it requires tissue biopsy, and generally these patients are unfit to undergo invasive lung biopsies. We describe a novel technique in a case with uncontrolled diabetes mellitus with nonresolving pulmonary cavitary disease where convex probe endobronchial ultrasound (EBUS)-guided aspiration of lung cavity wall showed classical histopathological picture establishing the diagnosis of mucorale infection. EBUS being real-time, minimally invasive technique with minimal risk of complications, led to early diagnosis, and prompt treatment. This appears to be a novel diagnostic modality in pulmonary mucormycosis with minimal complications as compared with other biopsy methods with very high complication risk.

Potential drug interaction with opioid agonist in the setting of chronic low-dose opioid antagonist use.

Low dose naltrexone (LDN) has been evaluated in several small studies for the treatment of inflammatory conditions. It is thought to work through modulation of inflammatory mediators and upregulation of endogenous opioid receptors. This may hypersensitize patients to exogenous opioids. Drug-drug interaction screening tools built into electronic health records and other services identify the interaction as risk of opioid withdrawal rather than hypersensitivity. We present a case of a drug-drug interaction in a patient who was receiving LDN treatment of multiple sclerosis. The patient received a single dose of oxycodone 5mg that resulted in obtundation unresponsive to painful stimuli necessitating the administration of naloxone boluses and infusion along with admission to the intensive care unit for 1 night. The patient responded well to naloxone therapy. He was discharged in satisfactory condition.

Pharmacological management of cerebral venous sinus thrombosis with full-dose IV heparin infusion and its clinical outcomes.

OBJECTIVE: To report a case of successful use of unfractionated heparin (UFH) infusion to treat cerebral venous sinus thrombosis (CVST). CASE SUMMARY: A 54-year-old female with a history of ovarian cancer addressed through palliative care, presents to the Emergency Department complaining of nausea, vomiting and headache for the last 72h. The patient was on a home regimen of enoxaparin 1.5mg/kg subcutaneously daily for recent pulmonary embolism and deep vein thrombosis that developed while on warfarin therapy previously. CT scan showed superior sagittal sinus thrombosis. UFH infusion was initiated and continued for 48h until the headache dissipated. DISCUSSION: Stable CVST may be treated with UFH infusion; however, there is limited literature that describes UFH dosing for CVST management. CONCLUSIONS: UFH may be considered as one of the pharmacological agents to manage CVST. The dosing for UFH bolus and infusion is similar to treatment dose for pulmonary embolism/deep vein thrombosis management with goal anti-Xa between 0.3 and 0.7units/mL.

RNA Strand Displacement Assay for Hepatitis E Virus Helicase.

The hepatitis E virus (HEV) helicase uses ATP to unwind the RNA duplexes. This is an essential step for viral replication. This protocol aims to measure the double strand RNA unwinding activity of the HEV helicase.

RNA-dependent RNA Polymerase Assay for Hepatitis E Virus.

RNA-dependent RNA polymerase (RdRp) is essential for the replication of viral RNA for RNA viruses. It synthesizes the complementary strand of viral genomic RNA, which is used subsequently as a template to generate more copies of viral genome. This assay measures activity of the hepatitis E virus (HEV) RdRp. In contrast to protocols available to assay the RdRp activity of many other viruses, this assay utilizes DIG-11-UTP as a nonradioactive alternative to 32P-UTP, thereby increasing the convenience of performing the assay.

Hemorrhagic sarcoid pleural effusion: A rare entity.

Involvement of pleura by sarcoidosis remains a rare manifestation and varies from pleural effusion, pneumothorax, pleural thickening, hydropneumothorax, trapped lung, hemothorax, or chylothorax. Sarcoid pleural effusions presenting as hemorrhagic effusions are even more rare. We report a case of active pulmonary sarcoidosis presenting as hemorrhagic pleural effusion requiring tissue diagnosis to rule out malignancy. The rarity of the presentation prompted us to report this case.