The rockefeller university

The present study investigated the role of oestrogen receptor (ER) in the ventromedial nucleus of the hypothalamus (VMN), the preoptic area (POA), the medial amygdala (MePD) and the bed nucleus of stria terminalis (BNST) in sociosexual behaviour in female rats. This was conducted in two sets of experiments, with the VMN and POA investigated in the first set, and the MePD and BNST in the second set. The VMN and POA received intense projections from the MePD and BNST. We used a short hairpin RNA encoded within an adeno-associated viral vector directed against the gene for ER to reduce the number of ER in the VMN or POA (first set of experiments) or in the BNST or MePD (second set of experiments) in female rats. The rats were housed in groups of four ovariectomised females and three males in a seminatural environment for 8days. Compared with traditional test set-ups, the seminatural environment provides an arena in which the rats can express their full behavioural repertoire, which allowed us to investigate multiple aspects of social and sexual behaviour in groups of rats. Behavioural observation was performed after oestrogen and progesterone injections. A reduction of ER expression in the VMN or POA diminished the display of paracopulatory behaviours and lordosis responses compared to controls, whereas the lordosis quotient remained unaffected. This suggests that ER in the VMN and POA play an important role in intrinsic sexual motivation. The reduction in ER did not affect the social behaviour of the females, although the males sniffed and pursued the females with reduced ER less than the controls. This suggests that the ER in the VMN and POA is involved in the regulation of sexual attractiveness of females. The ER in the MePD and BNST, on the other hand, plays no role in sociosexual behaviour.

Axon pathfinding is orchestrated by numerous guidance cues, including Slits and their Robo receptors, but it remains unclear how information from multiple cues is integrated or filtered. Robo3, a Robo family member, allows commissural axons to reach and cross the spinal cord midline by antagonizing Robo1/2-mediated repulsion from midline-expressed Slits and potentiating deleted in colorectal cancer (DCC)-mediated midline attraction to Netrin-1, but without binding either Slits or Netrins. We identified a secreted Robo3 ligand, neural epidermal growth factor-like-like 2 (NELL2), which repels mouse commissural axons through Robo3 and helps steer them to the midline. These findings identify NELL2 as an axon guidance cue and establish Robo3 as a multifunctional regulator of pathfinding that simultaneously mediates NELL2 repulsion, inhibits Slit repulsion, and facilitates Netrin attraction to achieve a common guidance purpose.

CagA is a multifunctional toxin of Helicobacter pylori that is secreted into host epithelial cells by a type IV secretion system. Following host cell translocation, CagA interferes with various host-cell signalling pathways. Most notably this toxin is involved in the disruption of apical-basolateral cell polarity and cell adhesion, as well as in the induction of cell proliferation, migration and cell morphological changes. These are processes that also play an important role in epithelial-to-mesenchymal transition and cancer cell invasion. In fact, CagA is considered as the only known bacterial oncoprotein. The cellular effects are triggered by a variety of CagA activities including the inhibition of serine-threonine kinase Par1b/MARK2 and the activation of tyrosine phosphatase SHP-2. Additionally, CagA was described to affect the activity of Src family kinases and C-terminal Src kinase (Csk) suggesting that interference with multiple cellular kinase- and phosphatase-associated signalling pathways is a major function of CagA. Here, we describe the effect of CagA on protein kinase C-related kinase 2 (PRK2), which acts downstream of Rho GTPases and is known to affect cytoskeletal rearrangements and cell polarity. CagA interacts with PRK2 and inhibits its kinase activity. Because PRK2 has been linked to cytoskeletal rearrangements and establishment of cell polarity, we suggest that CagA may hijack PRK2 to further manipulate cancer-related signalling pathways.

The protein-coding exome of a patient with a monogenic disease contains about 20,000 variants, only one or two of which are disease causing. We found that 58% of rare variants in the protein-coding exome of the general population are located in only 2% of the genes. Prompted by this observation, we aimed to develop a gene-level approach for predicting whether a given human protein-coding gene is likely to harbor disease-causing mutations. To this end, we derived the gene damage index (GDI): a genome-wide, gene-level metric of the mutational damage that has accumulated in the general population. We found that the GDI was correlated with selective evolutionary pressure, protein complexity, coding sequence length, and the number of paralogs. We compared GDI with the leading gene-level approaches, genic intolerance, and de novo excess, and demonstrated that GDI performed best for the detection of false positives (i.e., removing exome variants in genes irrelevant to disease), whereas genic intolerance and de novo excess performed better for the detection of true positives (i.e., assessing de novo mutations in genes likely to be disease causing).

We report the updated classification of primary immunodeficiencies compiled by the Primary Immunodeficiency Expert Committee (PID EC) of the International Union of Immunological Societies (IUIS). In the two years since the previous version, 34 new gene defects are reported in this updated version. For each disorder, the key clinical and laboratory features are provided. In this new version we continue to see the increasing overlap between immunodeficiency, as manifested by infection and/or malignancy, and immune dysregulation, as manifested by auto-inflammation, auto-immunity, and/or allergy. There is also an increased number of genetic defects that lead to susceptibility to specific organisms which reflects the finely tuned nature of immune defense systems. This classification is the most up to date catalogue of all known and published primary immunodeficiencies and acts as a current reference of the knowledge of these conditions and is an important aid for the genetic and molecular diagnosis of patients with these rare diseases.

The regulation of protein synthesis contributes to gene expression in both normal physiology and disease, yet kinetic investigations of the human translation mechanism are currently lacking. Using single-molecule fluorescence imaging methods, we have quantified the nature and timing of structural processes in human ribosomes during single-turnover and processive translation reactions. These measurements reveal that functional complexes exhibit dynamic behaviors and thermodynamic stabilities distinct from those observed for bacterial systems. Structurally defined sub-states of pre- and post-translocation complexes were sensitive to specific inhibitors of the eukaryotic ribosome, demonstrating the utility of this platform to probe drug mechanism. The application of three-color single-molecule fluorescence resonance energy transfer (smFRET) methods further revealed a long-distance allosteric coupling between distal tRNA binding sites within ribosomes bearing three tRNAs, which contributed to the rate of processive translation.

As nucleators of the mitotic spindle and primary cilium, centrosomes play crucial roles in equal segregation of DNA content to daughter cells, coordination of growth and differentiation, and transduction of homeostatic cues. Whereas the majority of mammalian cells carry no more than two centrosomes per cell, exceptions to this rule apply in certain specialized tissues and in select disease states, including cancer. Centrosome amplification, or the condition of having more than two centrosomes per cell, has been suggested to contribute to instability of chromosomes, imbalance in asymmetric divisions, and reorganization of tissue architecture; however, the degree to which these conditions are a direct cause of or simply a consequence of human disease is poorly understood. Here we addressed this issue by generating a mouse model inducing centrosome amplification in a naturally proliferative epithelial tissue by elevating Polo-like kinase 4 (Plk4) expression in the skin epidermis. By altering centrosome numbers, we observed multiciliated cells, spindle orientation errors, and chromosome segregation defects within developing epidermis. None of these defects was sufficient to impart a proliferative advantage within the tissue, however. Rather, impaired mitoses led to p53-mediated cell death and contributed to defective growth and stratification. Despite these abnormalities, mice remained viable and healthy, although epidermal cells with centrosome amplification were still appreciable. Moreover, these abnormalities were insufficient to disrupt homeostasis and initiate or enhance tumorigenesis, underscoring the powerful surveillance mechanisms in the skin.

Fibrolamellar hepatocellular carcinoma (FLHCC) tumors all carry a deletion of similar to 400 kb in chromosome 19, resulting in a fusion of the genes for the heat shock protein, DNAJ (Hsp40) homolog, subfamily B, member 1, DNAJB1, and the catalytic subunit of protein kinase A, PRKACA. The resulting chimeric transcript produces a fusion protein that retains kinase activity. No other recurrent genomic alterations have been identified. Here we characterize the molecular pathogenesis of FLHCC with transcriptome sequencing (RNA sequencing). Differential expression (tumor vs. adjacent normal tissue) was detected for more than 3,500 genes (log(2) fold change >= 1, false discovery rate <= 0.01), many of which were distinct from those found in hepatocellular carcinoma. Expression of several known oncogenes, such as ErbB2 and Aurora Kinase A, was increased in tumor samples. These and other dysregulated genes may serve as potential targets for therapeutic intervention.

The yeast homologs of the ATM and ATR DNA damage response kinases play key roles in telomerase-mediated telomere maintenance, but the role of ATM/ATR in the mammalian telomerase pathway has been less clear. Here, we demonstrate the requirement for ATM and ATR in the localization of telomerase to telomeres and telomere elongation in immortal human cells. Stalled replication forks increased telomerase recruitment in an ATR-dependent manner. Furthermore, increased telomerase recruitment was observed upon phosphorylation of the shelterin component TRF1 at an ATM/ATR target site (S367). This phosphorylation leads to loss of TRF1 from telomeres and may therefore increase replication fork stalling. ATM and ATR depletion reduced assembly of the telomerase complex, and ATM was required for telomere elongation in cells expressing POT1 Delta OB, an allele of POT1 that disrupts telomere-length homeostasis. These data establish that human telomerase recruitment and telomere elongation are modulated by DNA-damage-transducing kinases.

There are now nearly 300 single-gene inborn errors of immunity underlying phenotypes as diverse as infection, malignancy, allergy, auto-immunity, and auto-inflammation. For each of these five categories, a growing variety of phenotypes are ascribed to Primary Immunodeficiency Diseases (PID), making PIDs a rapidly expanding field of medicine. The International Union of Immunological Societies (IUIS) PID expert committee (EC) has published every other year a classification of these disorders into tables, defined by shared pathogenesis and/or clinical consequences. In 2013, the IUIS committee also proposed a more user-friendly, phenotypic classification, based on the selection of key phenotypes at the bedside. We herein propose the revised figures, based on the accompanying 2015 IUIS PID EC classification.