The rockefeller university

The ability to hide in the animal kingdom is essential for survival; the same is true for bacteria. Streptococcus pyogenes is considered one of the more successful stealth bacteria in its production of a hyaluronic acid capsule that is chemically identical to the hyaluronic acid lining human joints. It has also acquired the capacity to enter eukaryotic cells to avoid the onslaught of the host's immune defenses, as well as drugs. From this intracellular vantage point, it may remain dormant from days to weeks, only to cause disease again at a later time, perhaps causing a relapse in a drug-treated patient. We now learn that it is able to enter macrophages as well, enabling the Streptococcus to use this "Trojan horse" approach to be transported to distant sites in the body.

The neuropeptide PDF promotes the normal sequencing of circadian behavioral rhythms in Drosophila, but its signaling mechanisms are not well understood. We report daily rhythmicity in responsiveness to PDF in critical pacemakers called small LNvs. There is a daily change in potency, as great as 10-fold higher, around dawn. The rhythm persists in constant darkness and does not require endogenous ligand (PDF) signaling or rhythmic receptor gene transcription. Furthermore, rhythmic responsiveness reflects the properties of the pacemaker cell type, not the receptor. Dopamine responsiveness also cycles, in phase with that of PDF, in the same pacemakers, but does not cycle in large LNv. The activity of RalA GTPase in s-LNv regulates PDF responsiveness and behavioral locomotor rhythms. Additionally, cell-autonomous PDF signaling reversed the circadian behavioral effects of lowered RalA activity. Thus, RalA activity confers high PDF responsiveness, providing a daily gate around the dawn hours to promote functional PDF signaling.

The neuron specific RNA-binding proteins NOVA1 and NOVA2 are highly homologous alternative splicing regulators. NOVA proteins regulate at least 700 alternative splicing events in vivo, yet relatively little is known about the biologic consequences of NOVA action and in particular about functional differences between NOVA1 and NOVA2. Transcriptome-wide searches for isoform-specific functions, using NOVA1 and NOVA2 specific HITS-CLIP and RNA-seq data from mouse cortex lacking either NOVA isoform, reveals that NOVA2 uniquely regulates alternative splicing events of a series of axon guidance related genes during cortical development. Corresponding axonal pathfinding defects were specific to NOVA2 deficiency: Nova2-/- but not Nova1-/- mice had agenesis of the corpus callosum, and axonal outgrowth defects specific to ventral motoneuron axons and efferent innervation of the cochlea. Thus we have discovered that NOVA2 uniquely regulates alternative splicing of a coordinate set of transcripts encoding key components in cortical, brainstem and spinal axon guidance/outgrowth pathways during neural differentiation, with severe functional consequences in vivo.

The bacterial CRISPR/Cas9 system allows sequence-specific gene editing in many organisms and holds promise as a tool to generate models of human diseases, for example, in human pluripotent stem cells(1,2). CRISPR/Cas9 introduces targeted double-stranded breaks (DSBs) with high efficiency, which are typically repaired by non-homologous end-joining (NHEJ) resulting in nonspecific insertions, deletions or other mutations (indels)(2). DSBs may also be repaired by homology-directed repair (HDR)(1,2) using a DNA repair template, such as an introduced single-stranded oligo DNA nucleotide (ssODN), allowing knock-in of specific mutations(3). Although CRISPR/Cas9 is used extensively to engineer gene knockouts through NHEJ, editing by HDR remains inefficient(3-8) and can be corrupted by additional indels(9), preventing its widespread use for modelling genetic disorders through introducing disease-associated mutations. Furthermore, targeted mutational knock-in at single alleles to model diseases caused by heterozygous mutations has not been reported. Here we describe a CRISPR/Cas9-based genome-editing framework that allows selective introduction of mono-and bi-allelic sequence changes with high efficiency and accuracy. We show that HDR accuracy is increased dramatically by incorporating silent CRISPR/Casblocking mutations along with pathogenic mutations, and establish a method termed 'CORRECT' for scarless genome editing. By characterizing and exploiting a stereotyped inverse relationship between a mutation's incorporation rate and its distance to the DSB, we achieve predictable control of zygosity. Homozygous introduction requires a guide RNA targeting close to the intended mutation, whereas heterozygous introduction can be accomplished by distance-dependent suboptimal mutation incorporation or by use of mixed repair templates. Using this approach, we generated human induced pluripotent stem cells with heterozygous and homozygous dominant early onset Alzheimer's disease-causing mutations in amyloid precursor protein (APP(Swe))(10) and presenilin 1 (PSEN1M146V)(11) and derived cortical neurons, which displayed genotype-dependent disease-associated phenotypes. Our findings enable efficient introduction of specific sequence changes with CRISPR/Cas9, facilitating study of human disease.

A published review of the literature by Dutch investigators in 2004 suggested significant outcome differences between spontaneously - and in vitro fertilization (IVF) - conceived singleton and twin pregnancies. Here we review whether later studies between 2004-2015 confirmed these findings. Though methodologies of here reviewed studies varied, and all were retrospective, they overall confirmed results of the 2004 review, and supported significant outcome variances between spontaneously- and IVF-conceived pregnancies: IVF singletons demonstrate significantly poorer and IVF twins significantly better perinatal outcomes than spontaneously conceived singletons and twins, with differences stable over time, and with overall obstetrical outcomes significantly improved. Exaggerations of severe IVF twin risks are likely in the 50 % range, while exaggerations of milder perinatal risks are approximately in 25 % range. Though elective single embryo transfers (eSET) have been confirmed to reduce pregnancy chances, they are, nevertheless, increasingly utilized. eSET, equally unquestionably, however, reduces twin pregnancies. Because twin pregnancies have been alleged to increase outcome risks in comparison to singleton pregnancies, here reported findings should affect the ongoing discussion whether increased twin risks are factual. With no risk excess, eSET significantly reduces IVF pregnancy chances without compensatory benefits and, therefore, is not advisable in IVF, unless patients do not wish to conceive twins or have medical contraindications to conceiving twins.

RNA-binding proteins (RBPs) control multiple aspects of post-transcriptional gene regulation and function during various biological processes in the nervous system. To further reveal the functional significance of RBPs during neural development, we carried out an in vivo RNAi screen in the dorsal spinal cord interneurons, including the commissural neurons. We found that the NOVA family of RBPs play a key role in neuronal migration, axon outgrowth, and axon guidance. Interestingly, Nova mutants display similar defects as the knockout of the Dcc transmembrane receptor. We show here that Nova deficiency disrupts the alternative splicing of Dcc, and that restoring Dcc splicing in Nova knockouts is able to rescue the defects. Together, our results demonstrate that the production of DCC splice variants controlled by NOVA has a crucial function during many stages of commissural neuron development.

Tumors are composed of multiple cell types besides the tumor cells themselves, including innate immune cells such as macrophages. Tumor-associated macrophages (TAMs) are a heterogeneous population of myeloid cells present in the tumor microenvironment (TME). Here, they contribute to immunosuppression, enabling the establishment and persistence of solid tumors as well as metastatic dissemination. We have found that the pattern recognition scavenger receptor MARCO defines a subtype of suppressive TAMs and is linked to clinical outcome. An anti-MARCO monoclonal antibody was developed, which induces anti-tumor activity in breast and colon carcinoma, as well as in melanoma models through reprogramming-TAM-populations to a pro-inflammatory phenotype and increasing tumor immunogenicity. This anti-tumor activity is dependent on the inhibitory Fc-receptor, Fc gamma RIIB, and also enhances the efficacy of checkpoint therapy. These results demonstrate that immunotherapies using antibodies designed to modify myeloid cells of the TME represent a promising mode of cancer treatment.

L1 (also known as L1CAM) is a trans-membrane glycoprotein mediating neuron-neuron adhesion through homophilic and heterophilic interactions. Although experimental evidence has implicated L1 in axonal outgrowth, fasciculation and pathfinding, its contribution to voltage-gated Na+ channel function and membrane excitability has remained unknown. Here, we show that firing rate, single cell spiking frequency and Na+ current density are all reduced in hippocampal excitatory neurons from L1-deficient mice both in culture and in slices owing to an overall reduced membrane expression of Na+ channels. Remarkably, normal firing activity was restored when L1 was reintroduced into L1-deficient excitatory neurons, indicating that abnormal firing patterns are not related to developmental abnormalities, but are a direct consequence of L1 deletion. Moreover, L1 deficiency leads to impairment of action potential initiation, most likely due to the loss of the interaction of L1 with ankyrin G that produces the delocalization of Na+ channels at the axonal initial segment. We conclude that L1 contributes to functional expression and localization of Na+ channels to the neuronal plasma membrane, ensuring correct initiation of action potential and normal firing activity.