Rab8b GTPase, a protein transport regulator, is an interacting partner of otoferlin, defective in a human autosomal recessive deafness form

Mutations within OTOF encoding otoferlin lead to a recessive disorder called DFNB9. Several studies have indicated otoferlin's association with ribbon synapses of cochlear sensory hair cells, as well as data showing the protein's presence in neurons, nerve fibers and hair cells, suggesting a more ubiquitous function. Otoferlin's co-localization not only with ribbon synaptic proteins, but also with additional endosomal (EEA1) or Golgi proteins (GM130) were motivation for a search for further binding partners of otoferlin by a yeast two-hybrid screen in a rodent cochlear cDNA library (P3-P15). This screen identified Rab8b GTPase as a novel interacting partner, substantiated by transient co-expression and co-localization in HEK 293 cells and co-immunoprecipitation of the complex using tagged proteins in vitro and native proteins from cochlea. This finding implies that otoferlin could be a part of components contributing to trans-Golgi trafficking.     

A structural motif in the C-terminal tail of slo1 confers carbon monoxide sensitivity to human BKCa channels

Carbon monoxide (CO) is a potent activator of large conductance, calcium-dependent potassium (BK_Ca) channels of vascular myocytes and carotid body glomus cells or when heterologously expressed. Using the human BK_Ca channel α₁-subunit (hSlo1; KCNMA1) stably and transiently expressed in human embryonic kidney 293 cells, the mechanism and structural basis of channel activation by CO was investigated in inside–out, excised membrane patches. Activation by CO was concentration dependent (EC₅₀ ∼20 μM), rapid, reversible, and evoked a shift in the V_0.5 of −20 mV. CO evoked no changes in either single channel conductance or in deactivation rate but augmented channel activation rate. Activation was independent of the redox state of the channel, or associated compounds/protein partners, and was partially dependent on [Ca²⁺]_i in the physiological range (100–1,000 nM). Importantly, CO “super-stimulated” BK_Ca activity even in saturating [Ca²⁺]_i. Single or double mutation of two histidine residues previously implicated in CO sensing did not suppress CO activation but replacing the S9–S10 module of the C-terminal of Slo1 with that of Slo3 completely prevented the action of CO. These findings show that a motif in the S9–S10 part of the C-terminal is essential for CO activation and suggest that this gas transmitter activates the BK_Ca channel by redox-independent changes in gating.     

Freeze-dried heart valve scaffolds

In this study, structure and biomechanical properties of freeze-dried decellularized porcine pulmonary heart valves were investigated. Heart valves were dissected from porcine hearts. The tissues were decellularized and separated in three groups: (1) without lyoprotectant, (2) with 5% sucrose, and (3) with a mixture of 2.5% sucrose and 2.5% hydroxyl ethylene starch (HES), and then underwent freeze-drying. Freeze-drying in the absence of lyoprotectants caused an overall more disintegrated appearance of the histological architecture of the porcine valves, especially between the fibrosa and the ventricularis layers. Freeze-dried tissues with lyoprotectants have a looser network of collagen and elastic fibers with bigger pore sizes. Tissue freeze-dried in the absence of lyoprotecants had the largest pore sizes, whereas the tissue freeze-dried in the presence of protectants showed pores of intermediate sizes between the decellularized tissue and the unprotected freeze-dried samples. Tissue freeze-dried with sucrose alone displayed less porosity than tissue freeze-dried with the sucrose/HES mixture, whereas no significant differences in biomechanical properties were observed. Decellularization decreased the elastic modulus of artery tissue. The elastic modulus of freeze-dried tissue without protectants resembled that of decellularized tissue. The elastic modulus values of freeze-dried tissue stabilized by lyoprotectants were greater than those of decellularized tissue, but similar to those of native tissue.     

Head and neck paragangliomas: clinical and molecular genetic classification

Head and neck paragangliomas are tumors arising from specialized neural crest cells. Prominent locations are the carotid body along with the vagal, jugular, and tympanic glomus. Head and neck paragangliomas are slowly growing tumors, with some carotid body tumors being reported to exist for many years as a painless lateral mass on the neck. Symptoms depend on the specific locations. In contrast to paraganglial tumors of the adrenals, abdomen and thorax, head and neck paragangliomas seldom release catecholamines and are hence rarely vasoactive. Petrous bone, jugular, and tympanic head and neck paragangliomas may cause hearing loss. The internationally accepted clinical classifications for carotid body tumors are based on the Shamblin Class I-III stages, which correspond to postoperative permanent side effects. For petrous-bone paragangliomas in the head and neck, the Fisch classification is used. Regarding the molecular genetics, head and neck paragangliomas have been associated with nine susceptibility genes: NF1, RET, VHL, SDHA, SDHB, SDHC, SDHD, SDHAF2 (SDH5), and TMEM127. Hereditary HNPs are mostly caused by mutations of the SDHD gene, but SDHB and SDHC mutations are not uncommon in such patients. Head and neck paragangliomas are rarely associated with mutations of VHL, RET, or NF1. The research on SDHA, SDHAF2 and TMEM127 is ongoing. Multiple head and neck paragangliomas are common in patients with SDHD mutations, while malignant head and neck paraganglioma is mostly seen in patients with SDHB mutations. The treatment of choice is surgical resection. Good postoperative results can be expected in carotid body tumors of Shamblin Class I and II, whereas operations on other carotid body tumors and other head and neck paragangliomas frequently result in deficits of the cranial nerves adjacent to the tumors. Slow growth and the tendency of hereditary head and neck paragangliomas to be multifocal may justify less aggressive treatment strategies.     

Recurrent deletions and reciprocal duplications of 10q11.21q11.23 including CHAT and SLC18A3 are likely mediated by complex low-copy repeats

We report 24 unrelated individuals with deletions and 17 additional cases with duplications at 10q11.21q21.1 identified by chromosomal microarray analysis. The rearrangements range in size from 0.3 to 12 Mb. Nineteen of the deletions and eight duplications are flanked by large, directly oriented segmental duplications of >98% sequence identity, suggesting that nonallelic homologous recombination (NAHR) caused these genomic rearrangements. Nine individuals with deletions and five with duplications have additional copy number changes. Detailed clinical evaluation of 20 patients with deletions revealed variable clinical features, with developmental delay (DD) and/or intellectual disability (ID) as the only features common to a majority of individuals. We suggest that some of the other features present in more than one patient with deletion, including hypotonia, sleep apnea, chronic constipation, gastroesophageal and vesicoureteral refluxes, epilepsy, ataxia, dysphagia, nystagmus, and ptosis may result from deletion of the CHAT gene, encoding choline acetyltransferase, and the SLC18A3 gene, mapping in the first intron of CHAT and encoding vesicular acetylcholine transporter. The phenotypic diversity and presence of the deletion in apparently normal carrier parents suggest that subjects carrying 10q11.21q11.23 deletions may exhibit variable phenotypic expressivity and incomplete penetrance influenced by additional genetic and nongenetic modifiers.     

Inflammatory synovial fluid microenvironment drives primary human chondrocytes to actively take part in inflammatory joint diseases

The role of human chondrocytes in the pathogenesis of cartilage degradation in rheumatic joint diseases has presently gained increasing interest. An active chondrocyte participation in local inflammation may play a role in the initiation and progression of inflammatory joint diseases and in a disruption of cartilage repair mechanisms resulting in cartilage degradation. In the present study, we hypothesized that inflammatory synovial fluid triggers human chondrocytes to actively take part in inflammatory processes in rheumatic joint diseases. Primary human chondrocytes were incubated in synovial fluids gained from patients with rheumatoid arthritis, psoriasis arthritis and reactive arthritis. The detection of vital cell numbers was determined by using Casy Cell Counter System. Apoptosis was measured by Annexin-V and 7AAD staining. Cytokine and chemokine secretion was determined by a multiplex suspension array. Detection of vital cells showed a highly significant decrease in chondrocyte numbers. Flow cytometry demonstrated a significant increase in apoptotic chondrocytes after the incubation. An active secretion of cytokines such as MCP-1 and MIF by chondrocytes was observed. The inflammatory synovial fluid microenvironment mediates apoptosis and cell death of chondrocytes. Moreover, in terms of cytokine secretion, it also induces an active participation of chondrocytes in ongoing inflammation.