Mutations in KPTN Cause Macrocephaly,Neurodevelopmental Delay,and Seizures

The proper development of neuronal circuits during neuromorphogenesis and neuronal-network formation is critically dependent on a coordinated and intricate series of molecular and cellular cues and responses. Although the cortical actin cytoskeleton is known to play a key role in neuromorphogenesis, relatively little is known about the specific molecules important for this process. Using linkage analysis and whole-exome sequencing on samples from families from the Amish community of Ohio, we have demonstrated that mutations in KPTN, encoding kaptin, cause a syndrome typified by macrocephaly, neurodevelopmental delay, and seizures. Our immunofluorescence analyses in primary neuronal cell cultures showed that endogenous and GFP-tagged kaptin associates with dynamic actin cytoskeletal structures and that this association is lost upon introduction of the identified mutations. Taken together, our studies have identified kaptin alterations responsible for macrocephaly and neurodevelopmental delay and define kaptin as a molecule crucial for normal human neuromorphogenesis.     

Defective Presynaptic Choline Transport Underlies Hereditary Motor Neuropathy

The neuromuscular junction (NMJ) is a specialized synapse with a complex molecular architecture that provides for reliable transmission between the nerve terminal and muscle fiber. Using linkage analysis and whole-exome sequencing of DNA samples from subjects with distal hereditary motor neuropathy type VII, we identified a mutation in SLC5A7, which encodes the presynaptic choline transporter (CHT), a critical determinant of synaptic acetylcholine synthesis and release at the NMJ. This dominantly segregating SLC5A7 mutation truncates the encoded product just beyond the final transmembrane domain, eliminating cytosolic-C-terminus sequences known to regulate surface transporter trafficking. Choline-transport assays in both transfected cells and monocytes from affected individuals revealed significant reductions in hemicholinium-3-sensitive choline uptake, a finding consistent with a dominant-negative mode of action. The discovery of CHT dysfunction underlying motor neuropathy identifies a biological basis for this group of conditions and widens the spectrum of disorders that derive from impaired NMJ transmission. Our findings compel consideration of mutations in SLC5A7 or its functional partners in relation to unexplained motor neuronopathies.     

Factors affecting the persistence of drug-induced reprogramming of the cancer methylome

Aberrant DNA methylation is a critical feature of cancer. Epigenetic therapy seeks to reverse these changes to restore normal gene expression. DNA demethylating agents, including 5-aza-2′-deoxycytidine (DAC), are currently used to treat certain leukemias, and can sensitize solid tumors to chemotherapy and immunotherapy. However, it has been difficult to pin the clinical efficacy of these agents to specific demethylation events, and the factors that contribute to the durability of response remain largely unknown. Here we examined the genome-wide kinetics of DAC-induced DNA demethylation and subsequent remethylation after drug withdrawal in breast cancer cells. We find that CpGs differ in both their susceptibility to demethylation and propensity for remethylation after drug removal. DAC-induced demethylation was most apparent at CpGs with higher initial methylation levels and further from CpG islands. Once demethylated, such sites exhibited varied remethylation potentials. The most rapidly remethylating CpGs regained >75% of their starting methylation within a month of drug withdrawal. These sites had higher pretreatment methylation levels, were enriched in gene bodies, marked by H3K36me3, and tended to be methylated in normal breast cells. In contrast, a more resistant class of CpG sites failed to regain even 20% of their initial methylation after 3 months. These sites had lower pretreatment methylation levels, were within or near CpG islands, marked by H3K79me2 or H3K4me2/3, and were overrepresented in sites that become aberrantly hypermethylated in breast cancers. Thus, whereas DAC-induced demethylation affects both endogenous and aberrantly methylated sites, tumor-specific hypermethylation is more slowly regained, even as normal methylation promptly recovers. Taken together, these data suggest that the durability of DAC response is linked to its selective ability to stably reset at least a portion of the cancer methylome.     

Establishment of normal ranges of laser Doppler blood flow in autologous tissue transplants.

Over a 3-year period, 136 patients were monitored following free autologous tissue transplantation using a laser Doppler flowmeter linked to a computerized data-acquisition system. This monitoring system has indicated perfusion compromise in free flaps more rapidly than clinical observation alone. Most important, this has resulted in an increase in salvage rate from 50.0 to 82.4 percent. In addition, our overall success rate has increased from 92.6 to 97.8 percent since introducing this monitor clinically. Computerization also has facilitated the collection of data, which has enabled us to establish expected values for postoperative blood flow in several types of donor tissues used for microvascular reconstruction. Finally, this computerized monitoring system has relieved personnel from basing decisions on subjective data.     

Computed tomographic (CT) guided percutaneous fine-needle aspiration biopsy: the Yale experience

Fifty-one CT-guided percutaneous fine-needle aspiration biopsies (PFNAB) were performed on 46 consecutive patients over 15 months. Cytologies were obtained to identify primary or secondary malignancy in the abdomen, pelvis, retroperitoneum, bone, and paraspinal region. Adequate cytologic material was obtained in 50 of 51 biopsies. There were 29 true-positive, 0 false-positive, 12 true-negative, and two false-negative cases with an overall accuracy rate of 95 percent. There was one minor complication, mesenteric hemorrhage, which did not require transfusion. Fifteen of the 51 biopsies were performed on outpatients. The procedure is an accurate, safe, and cost-effective nonsurgical means of diagnosing primary or secondary malignancy.     

Growth responses of fish to changing environmental conditions in a South Carolina cooling reservoir

Synopsis Significant changes in growth of bluegills,Lepomis macrochirus, largemouth bass,Micropterus salmoides, and black crappies,Pomoxis nigromaculatus, were observed in Keowee Reservoir, South Carolina, during the 10 years (1968–1977) following impoundment. During this period the reservoir filled, water temperatures were increased by heated effluent from a nuclear power plant, and threadfin shad,Dorosoma petenense, were stocked. Growth was apparently regulated by abundance and size of prey eaten by fish; elevated water temperatures had no measurable effect.