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.

Endoplasmic Reticulum Stress Induced Synthesis of a Novel Viral Factor Mediates Efficient Replication of Genotype-1 Hepatitis E Virus.

Hepatitis E virus (HEV) causes acute hepatitis in many parts of the world including Asia, Africa and Latin America. Though self-limiting in normal individuals, it results in ~30% mortality in infected pregnant women. It has also been reported to cause acute and chronic hepatitis in organ transplant patients. Of the seven viral genotypes, genotype-1 virus infects humans and is a major public health concern in South Asian countries. Sporadic cases of genotype-3 and 4 infection in human and animals such as pigs, deer, mongeese have been reported primarily from industrialized countries. Genotype-5, 6 and 7 viruses are known to infect animals such as wild boar and camel, respectively. Genotype-3 and 4 viruses have been successfully propagated in the laboratory in mammalian cell culture. However, genotype-1 virus replicates poorly in mammalian cell culture and no other efficient model exists to study its life cycle. Here, we report that endoplasmic reticulum (ER) stress promotes genotype-1 HEV replication by inducing cap-independent, internal initiation mediated translation of a novel viral protein (named ORF4). Importantly, ORF4 expression and stimulatory effect of ER stress inducers on viral replication is specific to genotype-1. ORF4 protein sequence is mostly conserved among genotype-1 HEV isolates and ORF4 specific antibodies were detected in genotype-1 HEV patient serum. ORF4 interacted with multiple viral and host proteins and assembled a protein complex consisting of viral helicase, RNA dependent RNA polymerase (RdRp), X, host eEF1α1 (eukaryotic elongation factor 1 isoform-1) and tubulinß. In association with eEF1α1, ORF4 stimulated viral RdRp activity. Furthermore, human hepatoma cells that stably express ORF4 or engineered proteasome resistant ORF4 mutant genome permitted enhanced viral replication. These findings reveal a positive role of ER stress in promoting genotype-1 HEV replication and pave the way towards development of an efficient model of the virus.

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.

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.

Percutaneous closure of the left atrial appendage for stroke prevention in atrial fibrillation: an alternative to lifelong anticoagulation?

Atrial fibrillation is an important risk factor for thromboembolic stroke and it significantly increases the risk of stroke. The left atrial appendage (LAA) is the most common site of thrombus formation in nonvalvular atrial fibrillation, and the recent applications of percutaneous LAA closure devices offer a promising alternative for patients who are unable to tolerate lifelong anticoagulation. Critical care nurses who understand the procedures and are familiar with the various devices used for LAA closure will be well prepared to provide optimum care and appropriate education for these patients.

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.

Rotational lamellar scleral flap for the management of posttrabeculectomy bleb leak.

We describe a technique of rotational lamellar scleral flap for surgical repair in cases of posttrabeculectomy aqueous leak in patients with button holing or necrosis of the trabeculectomy flap. A rotational scleral flap is marked out from the sclera adjacent to the trabeculectomy site followed by a lamellar dissection to fashion the flap. Relaxing cuts are made at the base of the flap so as to ensure that the rotation flap adequately covers the site of aqueous leak. This flap is then secured to the underlying sclera and the cornea at the limbus.

HIV medication therapy management services in community pharmacies.

OBJECTIVES: To present a rationale and a proposed structure to support pharmacist-delivered medication therapy management (MTM) for human immunodeficiency virus (HIV ) disease and to outline challenges to implementing and sustaining the service. DATA SOURCES: Professional literature. SUMMARY: Historically, the effect of pharmacy services for HIV-infected persons has been demonstrated in inpatient and clinic-based settings. Developing similar programs adapted for community pharmacists could be a model of care to improve patient adherence to antiretroviral therapy and retention in care. Initiation of antiretroviral therapy and regular monitoring of CD4+ cell count, HIV RNA viral load, adverse drug events, and adherence form the backbone of successful medical management of HIV infection. Support for these services can be provided to HIV-infected patients through pharmacist-managed HIV MTM programs in community pharmacy settings in collaboration with primary providers and other health care professionals. CONCLUSION: Community pharmacists can help meet the growing need for HIV care through provision of MTM services. Although resources have been developed, including the general MTM framework, challenges of adequate training, education, and support of community pharmacists need to be addressed in order for HIV MTM to be a successful model.

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.

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.

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.

Cataract surgery in the setting of trauma.

PURPOSE: To review the changes in the surgical techniques used for cataract removal in the setting of trauma and their postoperative outcome. RECENT FINDINGS: Primary cataract removal with intraocular lens (IOL) implantation is the commonly followed procedure for penetrating injuries with cataract. IOL implantation has evolved through various techniques namely 'in the bag', 'in the sulcus', epilenticular implantation, anterior chamber IOL, scleral fixated IOL and recently glued IOL. SUMMARY: Certain lacerating injuries of the anterior segment are particularly amenable to cataract extraction and IOL implantation at the time of primary laceration repair. This approach obviates additional operative and anesthetic risks, while affording timelier visual rehabilitation. Secondary lens removal may also be indicated in cases of severe corneal injury and marked edema, which may interfere with intraocular visualization.

Anterior segment transplantation with a novel biosynthetic graft.

PURPOSE: Staphylectomy with sclerokeratoplasty has been performed in cases with large anterior staphyloma. This report describes a technique using a new biosynthetic graft to simulate the anterior segment in the eye of a 4-month-old child with diffuse anterior staphyloma and cataractous lens with compromised zonules. METHODS: The graft had a biologic part (corneoscleral button) and a synthetic part (aniridia intraocular lens). Two partial thickness flaps and sclerotomies were made in the graft through which the lens haptics were externalized. After staphylectomy and lensectomy of the host, the graft was sutured. After intrascleral tuck of the lens haptics before fibrin glue-assisted flap closure was then done. RESULTS: On the first postoperative day, the child was following light with the operated eye, and closure of the lids and cosmesis were markedly improved. Between the first and fourth month follow-up, and with the normal eye occluded, the child was following light and reaching out for objects. The cornea was clear with no evidence of rejection or failure. At 5 months, a persistent epithelial defect developed, leading by 6 months into a nebulomacular opacity partially obscuring the pupil. At 6 months, the results of the technique are anatomically and cosmetically satisfactory; however, a longer follow-up term will establish the visual benefits. CONCLUSIONS: This technique may provide a suitable cosmetic and anatomic alternative to conventional procedures like staphylectomy with sclerokeratoplasty. It also retains the potential for more effective visual rehabilitation as an intraocular lens has been implanted.

Bilateral spontaneous in-the-bag anterior subluxation of PCIOL managed with glued IOL technique: A case report.

OBJECTIVE: Management of in-the-bag spontaneous bilateral subluxation of posterior-chamber intraocular lens(PCIOL) with sutureless fibrin-glue-assisted PCIOL implantation. METHODS: A patient of retinitis pigmentosa with spontaneous bilateral anterior in-the-bag subluxation of PCIOL was managed by IOL explantation followed by fibrin-glue-assisted sutureless PCIOL implantation. Two partial thickness limbal-based scleral flaps were created about 1.5 mm from the limbus under which sclerotomies were made. Intraocular lens explantation along with capsular bag was performed through the corneo-scleral tunnel incision. Single-piece rigid polymethylmethacrylate 6.5-mm optic IOL was introduced through the limbal wound with a McPherson forceps, both the IOL haptics were externalized under the scleral flap. The haptic ends were tucked in the scleral tunnel made with the 26G needle. Scleral flaps and the conjunctiva were closed with the fibrin glue. RESULTS: Preoperative best corrected visual acuity was 20/80 in the right and 20/120 in the left eye. Patient gained a best corrected visual acuity of 20/30 in both the eyes, with a bilateral stable PCIOL and clear cornea. CONCLUSIONS: Severe capsular contracture causing in-the-bag IOL subluxation in retinitis pigmentosa can be effectively managed with this new technique of sutureless fibrin-glue-assisted PCIOL implantation.

Femtosecond-assisted lamellar keratoplasty in atypical Avellino corneal dystrophy of Indian origin.

PURPOSE: To report a case of Avellino corneal dystrophy (ACD) in a patient of Indian origin treated with femtosecond-assisted lamellar keratoplasty (FALK). METHODS: A 6-year-old male patient presented with severe photophobia with decreased vision for 2 months. A clinical diagnosis of Avellino dystrophy was made after complete examination under anesthesia and FALK was performed. RESULTS: The postoperative period was uneventful with good symptomatic improvement and graft clarity. Histopathological study with special staining, namely Masson trichrome and Congo red stain, of the patient's corneal button showed features of both granular and lattice lesions suggestive of ACD. Genetic analysis showed absence of R124H mutation in BIGH3 gene. No recurrence or exacerbation was noted at 19-month follow-up. CONCLUSIONS: To our knowledge, this is the first case report of clinical, histopathological, microscopic features of ACD in young patient of Indian origin with absence of BIGH3 gene treated with FALK with IntraLase Femtosecond Laser for donor and recipient cuts.