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Accumulating evidence suggests that cerebellar dysfunction early in life is associated with autism spectrum disorder (ASD), but the molecular mechanisms underlying the cerebellar deficits at the cellular level are unclear. Tuberous sclerosis complex (TSC) is a neurocutaneous disorder that often presents with ASD. Here, we developed a cerebellar Purkinje cell (PC) model of TSC with patient-derived human induced pluripotent stem cells (hiPSCs) to characterize the molecular mechanisms underlying cerebellar abnormalities in ASD and TSC. Our results show that hiPSC-derived PCs from patients with pathogenic TSC2 mutations displayed mTORC1 pathway hyperactivation, defects in neuronal differentiation and RNA regulation, hypoexcitability and reduced synaptic activity when compared with those derived from controls. Our gene expression analyses revealed downregulation of several components of fragile X mental retardation protein (FMRP) targets in TSC2-deficient hiPSC-PCs. We detected decreased expression of FMRP, glutamate receptor 62 (GRID2), and pre- and post-synaptic markers such as synaptophysin and PSD95 in the TSC2-deficient hiPSC-PCs. The mTOR inhibitor rapamycin rescued the deficits in differentiation, synaptic dysfunction, and hypoexcitability of TSC2 mutant hiPSC-PCs in vitro. Our findings suggest that these gene expression changes and cellular abnormalities contribute to aberrant PC function during development in TSC affected individuals.

Zika virus (ZIKV) causes severe neurologic complications and fetal aberrations. Vaccine development is hindered by potential safety concerns due to antibody cross-reactivity with dengue virus and the possibility of disease enhancement. In contrast, passive administration of anti-ZIKV antibodies engineered to prevent enhancement may be safe and effective. Here, we report on human monoclonal antibody Z021, a potent neutralizer that recognizes an epitope on the lateral ridge of the envelope domain III (EDIII) of ZIKV and is protective against ZIKV in mice. When administered to macaques undergoing a high-dose ZIKV challenge, a single anti-EDIII antibody selected for resistant variants. Co-administration of two antibodies, Z004 and Z021, which target distinct sites on EDIII, was associated with a delay and a 3-to 4-log decrease in peak viremia. Moreover, the combination of these antibodies engineered to avoid enhancement prevented viral escape due to mutation in macaques, a natural host for ZIKV.

Class switch recombination (CSR) is a DNA recombination reaction that diversifies the effector component of antibody responses. CSR is initiated by activation-induced cytidine deaminase (AID), which targets transcriptionally active immunoglobulin heavy chain (Igh) switch donor and acceptor DNA. The 3' Igh super-enhancer, 3' regulatory region (3'RR), is essential for acceptor region transcription, but how this function is regulated is unknown. Here, we identify the chromatin reader ZMYND8 as an essential regulator of the 3'RR. In B cells, ZMYND8 binds promoters and super-enhancers, including the Igh enhancers. ZMYND8controls the 3'RR activity by modulating the enhancer transcriptional status. In its absence, there is increased 3'RR polymerase loading and decreased acceptor region transcription and CSR. In addition to CSR, ZMYND8 deficiency impairs somatic hypermutation (SHM) of Igh, which is also dependent on the 3'RR. Thus, ZMYND8 controls Igh diversification in mature B lymphocytes by regulating the activity of the 3' Igh super-enhancer.

Some of the most common infectious diseases are caused by bacteria that naturally colonise humans asymptomatically. Combating these opportunistic pathogens requires an understanding of the traits that differentiate infecting strains from harmless relatives. Staphylococcus epidermidis is carried asymptomatically on the skin and mucous membranes of virtually all humans but is a major cause of nosocomial infection associated with invasive procedures. Here we address the underlying evolutionary mechanisms of opportunistic pathogenicity by combining pangenome-wide association studies and laboratory microbiology to compare S. epidermidis from bloodstream and wound infections and asymptomatic carriage. We identify 61 genes containing infection-associated genetic elements (k-mers) that correlate with in vitro variation in known pathogenicity traits (biofilm formation, cell toxicity, interleukin-8 production, methicillin resistance). Horizontal gene transfer spreads these elements, allowing divergent clones to cause infection. Finally, Random Forest model prediction of disease status (carriage vs. infection) identifies pathogenicity elements in 415 S. epidermidis isolates with 80% accuracy, demonstrating the potential for identifying risk genotypes pre-operatively.

This article presents methods for generating in vitro fibrin clots and analyzing the effect of beta-amyloid (A beta) protein on clot formation and structure by spectrometry and scanning electron microscopy (SEM). A beta, which forms neurotoxic amyloid aggregates in Alzheimer's disease (AD), has been shown to interact with fibrinogen. This A beta-fibrinogen interaction makes the fibrin clot structurally abnormal and resistant to fibrinolysis. A beta-induced abnormalities in fibrin clotting may also contribute to cerebrovascular aspects of the AD pathology such as microinfarcts, inflammation, as well as, cerebral amyloid angiopathy (CAA). Given the potentially critical role of neurovascular deficits in AD pathology, developing compounds which can inhibit or lessen the A beta-fibrinogen interaction has promising therapeutic value. In vitro methods by which fibrin clot formation can be easily and systematically assessed are potentially useful tools for developing therapeutic compounds. Presented here is an optimized protocol for in vitro generation of the fibrin clot, as well as analysis of the effect of A beta and A beta-fibrinogen interaction inhibitors. The clot turbidity assay is rapid, highly reproducible and can be used to test multiple conditions simultaneously, allowing for the screening of large numbers of A beta-fibrinogen inhibitors. Hit compounds from this screening can be further evaluated for their ability to ameliorate A beta-induced structural abnormalities of the fibrin clot architecture using SEM. The effectiveness of these optimized protocols is demonstrated here using TDI-2760, a recently identified A beta-fibrinogen interaction inhibitor.

Female Aedes aegypti mosquitoes infect more than 400 million people each year with dangerous viral pathogens including dengue, yellow fever, Zika and chikungunya. Progress in understanding the biology of mosquitoes and developing the tools to fight them has been slowed by the lack of a high-quality genome assembly. Here we combine diverse technologies to produce the markedly improved, fully re-annotated AaegL5 genome assembly, and demonstrate how it accelerates mosquito science. We anchored physical and cytogenetic maps, doubled the number of known chemosensory ionotropic receptors that guide mosquitoes to human hosts and egg-laying sites, provided further insight into the size and composition of the sex-determining M locus, and revealed copy-number variation among glutathione S-transferase genes that are important for insecticide resistance. Using high-resolution quantitative trait locus and population genomic analyses, we mapped new candidates for dengue vector competence and insecticide resistance. AaegL5 will catalyse new biological insights and intervention strategies to fight this deadly disease vector.

Monotherapy of HIV-1 infection with single antiretroviral agents is ineffective because error-prone HIV-1 replication leads to the production of drug-resistant viral variants(1,2). Combinations of drugs can establish long-term control, however, antiretroviral therapy (ART) requires daily dosing, can cause side effects and does not eradicate the infection(3,4). Although anti-HIV-1 antibodies constitute a potential alternative to ART(5,6), treatment of viremic individuals with a single antibody also results in emergence of resistant viral variants(7-9). Moreover, combinations of first-generation anti-HIV-1 broadly neutralizing antibodies (bNAbs) had little measurable effect on the infection(10-12). Here we report on a phase 1b clinical trial (NCT02825797) in which two potent bNAbs, 3BNC117(13) and 10-1074(14), were administered in combination to seven HIV-1 viremic individuals. Infusions of 30 mg kg(-1) of each of the antibodies were well-tolerated. In the four individuals with dual antibody-sensitive viruses, immunotherapy resulted in an average reduction in HIV-1 viral load of 2.05 log(10) copies per ml that remained significantly reduced for three months following the first of up to three infusions. In addition, none of these individuals developed resistance to both antibodies. Larger studies will be necessary to confirm the efficacy of antibody combinations in reducing HIV-1 viremia and limiting the emergence of resistant viral variants.

Many hurdles have plagued the development of an effective vaccine for hepatitis C virus. In this issue of Cell Host & Microbe, Kinchen et al. (2018) and Flyak et al. (2018) report on the characterization of neutralizing antibodies from individuals that spontaneously cleared infection, providing insights that promise to propel vaccine design forward.

In hyper-IgE syndromes (HIES), a group of primary immunodeficiencies clinically overlapping with atopic dermatitis, early diagnosis is crucial to initiate appropriate therapy and prevent irreversible complications. Identification of underlying gene defects such as in DOCKS and STAT3 and corresponding molecular testing has improved diagnosis.Yet, in a child and her newborn sibling with HIES phenotype molecular diagnosis was misleading. Extensive analyses driven by the clinical phenotype identified an intronic homozygous DOCKS variant c.4626 76A > G creating a novel splice site as disease-causing. While the affected newborn carrying the homozygous variant had no expression of DOCK8 protein, in the index patient molecular diagnosis was compromised due to expression of altered and wildtype DOCKS transcripts and DOCK8 protein as well as defective STAT3 signaling. Sanger sequencing of lymphocyte subsets revealed that somatic alterations and reversions revoked the predominance of the novel over the canonical splice site in the index patient explaining DOCK8 protein expression, whereas defective STAT3 responses in the index patient were explained by a T cell phenotype skewed towards central and effector memory T cells. Hence, somatic alterations and skewed immune cell phenotypes due to selective pressure may compromise molecular diagnosis and need to be considered with unexpected clinical and molecular findings.

Combination antiretroviral therapy controls but does not cure HIV-1 infection because a small fraction of cells harbor latent viruses that can produce rebound viremia when therapy is interrupted. The circulating latent virus reservoir has been documented by a variety of methods, most prominently by viral outgrowth assays (VOAs) in which CD4(+) T cells are activated to produce virus in vitro, or more recently by amplifying proviral near full-length (NFL) sequences from DNA. Analysis of samples obtained in clinical studies in which individuals underwent analytical treatment interruption (ATI), showed little if any overlap between circulating latent viruses obtained from outgrowth cultures and rebound viruses from plasma. To determine whether intact proviruses amplified from DNA are more closely related to rebound viruses than those obtained from VOAs, we assayed 12 individuals who underwent ATI after infusion of a combination of two monoclonal anti-HIV-1 antibodies. A total of 435 intact proviruses obtained by NFL sequencing were compared with 650 latent viruses from VOAs and 246 plasma rebound viruses. Although, intact NFL and outgrowth culture sequences showed similar levels of stability and diversity with 39% overlap, the size of the reservoir estimated from NFL sequencing was larger than and did not correlate with VOAs. Finally, intact proviruses documented by NFL sequencing showed no sequence overlap with rebound viruses; however, they appear to contribute to recombinant viruses found in plasma during rebound.