In vitro higher-order oligomeric assembly of the respiratory syncytial virus M2-1 protein with longer RNAs.

The respiratory syncytial virus (RSV) M2-1 protein is a transcriptional antitermination factor crucial for efficiently synthesizing multiple full-length viral mRNAs. During RSV infection, M2-1 exists in a complex with mRNA within cytoplasmic compartments called inclusion body-associated granules (IBAGs). Prior studies showed that M2-1 can bind along the entire length of viral mRNAs instead of just gene-end (GE) sequences, suggesting that M2-1 has more sophisticated RNA recognition and binding characteristics. Here, we analyzed the higher oligomeric complexes formed by M2-1 and RNAs in vitro using size exclusion chromatography (SEC), electrophoretic mobility shift assays (EMSA), negative stain electron microscopy (EM), and mutagenesis. We observed that the minimal RNA length for such higher oligomeric assembly is about 14 nucleotides for polyadenine sequences, and longer RNAs exhibit distinct RNA-induced binding modality to M2-1, leading to enhanced particle formation frequency and particle homogeneity as the local RNA concentration increases. We showed that particular cysteine residues of the M2-1 cysteine-cysteine-cystine-histidine (CCCH) zinc-binding motif are essential for higher oligomeric assembly. Furthermore, complexes assembled with long polyadenine sequences remain unaffected when co-incubated with ribonucleases or a zinc chelation agent. Our study provided new insights into the higher oligomeric assembly of M2-1 with longer RNA.IMPORTANCERespiratory syncytial virus (RSV) causes significant respiratory infections in infants, the elderly, and immunocompromised individuals. The virus forms specialized compartments to produce genetic material, with the M2-1 protein playing a pivotal role. M2-1 acts as an anti-terminator in viral transcription, ensuring the creation of complete viral mRNA and associating with both viral and cellular mRNA. Our research focuses on understanding M2-1's function in viral mRNA synthesis by modeling interactions in a controlled environment. This approach is crucial due to the challenges of studying these compartments in vivo. Reconstructing the system in vitro uncovers structural and biochemical aspects and reveals the potential functions of M2-1 and its homologs in related viruses. Our work may contribute to identifying targets for antiviral inhibitors and advancing RSV infection treatment.

Left thoracotomy vs full sternotomy for centrifugal durable LVAD implantation: 1-year outcome comparison post-LVAD and post-heart transplantation.

Left ventricular assist device (LVAD) implantations have traditionally been approached through a full median sternotomy (FS). Recently, a minimally invasive left thoracotomy (LT) approach has been popularized. This study sought to compare the outcomes of FS and LT patients post-primary LVAD implantation and post-subsequent heart transplant (HT). This was a single-center retrospective study. 83 patients who underwent primary centrifugal durable LVAD implantation from January 2014 to June 2018 were included (FS, n = 41; LT, n = 42). 41 patients had a subsequent HT (FS, n = 19; LT, n = 22). Pre-operative patient demographics, intraoperative variables, post-operative 1-year survival, length of hospital stay, complications, and outcomes for LVAD implantation and following HT were analyzed. Intraoperative data showed that the LT group had a 23.4% longer mean LVAD implant surgical time (p < 0.01). One-year post-LVAD survival was similar between the two groups (p = 0.05). Complication rates, with the exception of the rate of hemorrhagic stroke (p = 0.04) post-LVAD implant were similar. One-year survival post-HT was similar between groups (p = 0.35). Complication rates and mean length of hospital stay were also similar (p = 1.0) post-HT. Our study demonstrated that LT approach does not negatively affect post-LVAD implantation or post-HT outcomes. Further, larger studies may determine more detailed effects of LT approach.

Optimal Conditions for In Vitro Assembly of Respiratory Syncytial Virus Nucleocapsid-like Particles.

The nucleocapsids (NCs) of the respiratory syncytial virus (RSV) can display multiple morphologies in vivo, including spherical, asymmetric, and filamentous conformations. Obtaining homogeneous ring-like oligomers in vitro is significant since they structurally represent one turn of the characteristic RSV NC helical filament. Here, we analyzed and optimized conditions for forming homogenous, recombinant nucleocapsid-like particles (NCLPs) of RSV in vitro. We examined the effects of modifying the integrated RNA length and sequence, altering incubation time, and varying buffer parameters, including salt concentration and pH, on ring-like NCLPs assembly using negative stain electron microscopy (EM) imaging. We showed that high-quality, homogeneous particles are assembled when incubating short, adenine-rich RNA sequences with RNA-free N associated with P (N0P). Further, we reported that a co-incubation duration greater than 3 days, a NaCl concentration between 100 mM and 200 mM, and a pH between 7 and 8 are optimal for N-RNA ring assembly with polyadenine RNA sequences. We believe assembling high-quality, homogeneous NCLPs in vitro will allow for further analysis of RSV RNA synthesis. This work may also lend insights into obtaining high-resolution nucleocapsid homogeneous structures for in vitro analysis of antiviral drug candidates against RSV and related viruses.

Mouse models and induced pluripotent stem cells in researching psychiatric disorders.

Psychiatric disorders are a problem for society both on a micro level involving patients and their families as well as on a macro level involving global economic costs. For years, scientists have relied on mouse models for research, but these have shortcomings that greatly hinder efforts to understand the pathophysiology and genetic factors of psychiatric disorders. Induced pluripotent stem cells (iPSCs) have shown potential to overcome obstacles that mouse models face and can provide patient-specific cells that allow for better understanding of genetic effects on psychiatric disorders. This review explores the current progress using iPSCs to model psychiatric disorders, specifically bipolar disorder and schizophrenia, while discussing remaining issues with iPSC use and how these issues can be resolved in the future.

A Novel Closed-Head Model of Mild Traumatic Brain Injury Using Focal Primary Overpressure Blast to the Cranium in Mice.

Mild traumatic brain injury (TBI) from focal head impact is the most common form of TBI in humans. Animal models, however, typically use direct impact to the exposed dura or skull, or blast to the entire head. We present a detailed characterization of a novel overpressure blast system to create focal closed-head mild TBI in mice. A high-pressure air pulse limited to a 7.5 mm diameter area on the left side of the head overlying the forebrain is delivered to anesthetized mice. The mouse eyes and ears are shielded, and its head and body are cushioned to minimize movement. This approach creates mild TBI by a pressure wave that acts on the brain, with minimal accompanying head acceleration-deceleration. A single 20-psi blast yields no functional deficits or brain injury, while a single 25-40 psi blast yields only slight motor deficits and brain damage. By contrast, a single 50-60 psi blast produces significant visual, motor, and neuropsychiatric impairments and axonal damage and microglial activation in major fiber tracts, but no contusive brain injury. This model thus reproduces the widespread axonal injury and functional impairments characteristic of closed-head mild TBI, without the complications of systemic or ocular blast effects or head acceleration that typically occur in other blast or impact models of closed-skull mild TBI. Accordingly, our model provides a simple way to examine the biomechanics, pathophysiology, and functional deficits that result from TBI and can serve as a reliable platform for testing therapies that reduce brain pathology and deficits.