Haploinsufficiency of ARID1B, a member of the SWI/SNF-a chromatin-remodeling complex, is a frequent cause of intellectual disability

Intellectual disability (ID) is a clinically and genetically heterogeneous common condition that remains etiologically unresolved in the majority of cases. Although several hundred diseased genes have been identified in X-linked, autosomal-recessive, or syndromic types of ID, the establishment of an etiological basis remains a difficult task in unspecific, sporadic cases. Just recently, de novo mutations in SYNGAP1, STXBP1, MEF2C, and GRIN2B were reported as relatively common causes of ID in such individuals. On the basis of a patient with severe ID and a 2.5 Mb microdeletion including ARID1B in chromosomal region 6q25, we performed mutational analysis in 887 unselected patients with unexplained ID. In this cohort, we found eight (0.9%) additional de novo nonsense or frameshift mutations predicted to cause haploinsufficiency. Our findings indicate that haploinsufficiency of ARID1B, a member of the SWI/SNF-A chromatin-remodeling complex, is a common cause of ID, and they add to the growing evidence that chromatin-remodeling defects are an important contributor to neurodevelopmental disorders.     

A chemical proteomics approach to identify c-di-GMP binding proteins in Pseudomonas aeruginosa

In many bacteria, high levels of the ubiquitous second messenger c-di-GMP have been demonstrated to suppress motility and to promote the establishment of surface-adherent biofilm communities. While molecular mechanisms underlying the synthesis and degradation of c-di-GMP have been comprehensively characterized, little is known about how c-di-GMP mediates its regulatory effects. In this study, we have established a chemical proteomics approach to identify c-di-GMP interacting proteins in the opportunistic pathogen Pseudomonas aeruginosa. A functionalized c-di-GMP analog, 2′-aminohexylcarbamoyl-c-di-GMP (2′-AHC-c-di-GMP), was chemically synthesized and following its immobilization used to perform affinity pull down experiments. Enriched proteins were subsequently identified by high-resolution mass spectrometry. 2′-AHC-c-di-GMP was also employed in surface plasmon resonance studies to evaluate and quantify the interaction of c-di-GMP with its potential target molecules in vitro. The biochemical tools presented here may serve the identification of novel classes of c-di-GMP effectors and thus contribute to a better characterization and understanding of the complex c-di-GMP signaling network.     

Comparative proteomics to evaluate multi drug resistance in Escherichia coli

Drug resistance in food-borne bacterial pathogens is an almost inevitable consequence of the use of antimicrobial drugs, used either therapeutically or to avoid infections in food-producing animals. In the past decades, the spread and inappropriate use of antibiotics have caused a considerable increase of antibiotics to which bacteria have developed resistance and, moreover, bacteria are becoming resistant to more than one antibiotic simultaneously. Understanding mechanisms at the molecular level is extremely important to control multi-resistant strains and to develop new therapeutic strategies. In the present study, comparative proteomics was applied to characterize membrane and cytosolic proteome in order to investigate the regulation of protein expression in multi-resistance E. coli isolated from young never vaccinated water buffalo. Results highlighted differentially expressed proteins under multi drug resistance conditions giving new insights about mechanisms involved in resistance, as quorum sensing mechanisms, and suggesting possible novel bacterial targets to develop alternative antibiotic drugs.     

The membrane-proximal domain of A Disintegrin and Metalloprotease 17 (ADAM17) is responsible for recognition of the interleukin-6 receptor and interleukin-1 receptor II

A great number of physiological processes are regulated by the release of ectodomains of membrane proteins. A Disintegrin And Metalloprotease 17 (ADAM17) is one of the important enzymes, which mediate this process called shedding. Today, more than 70 substrates of this transmembrane metalloprotease are known. This broad spectrum raises the question how ADAM17 recognizes its substrates specifically. Differently tagged ADAM17 deletion variants were used to demonstrate that exclusively the extracellular domains of ADAM17 are needed for interaction with two of its substrates, the IL-6R and the IL-1RII; whereas the transmembrane- and cytoplasmic-region are dispensable for this process. In the extracellular part solely the membrane-proximal domain of ADAM17 is mandatory for recognition of the two type-I transmembrane proteins, but not for the interaction with the type-II transmembrane molecule TNF-α.     

Co-Culture of S. epidermidis and Human Osteoblasts on Implant Surfaces: An Advanced In Vitro Model for Implant-Associated Infections

Objectives

Total joint arthroplasty is one of the most frequent and effective surgeries today. However, despite improved surgical techniques, a significant number of implant-associated infections still occur. Suitable in vitro models are needed to test potential approaches to prevent infection. In the present study, we aimed to establish an in vitro co-culture setup of human primary osteoblasts and S. epidermidis to model the onset of implant-associated infections, and to analyze antimicrobial implant surfaces and coatings.

Materials and Methods

For initial surface adhesion, human primary osteoblasts (hOB) were grown for 24 hours on test sample discs made of polystyrene, titanium alloy Ti6Al4V, bone cement PALACOS R^®, and PALACOS R^® loaded with antibiotics. Co-cultures were performed as a single-species infection on the osteoblasts with S. epidermidis (multiplicity of infection of 0.04), and were incubated for 2 and 7 days under aerobic conditions. Planktonic S. epidermidis was quantified by centrifugation and determination of colony-forming units (CFU). The quantification of biofilm-bound S. epidermidis on the test samples was performed by sonication and CFU counting. Quantification of adherent and vital primary osteoblasts on the test samples was performed by trypan-blue staining and counting. Scanning electron microscopy was used for evaluation of topography and composition of the species on the sample surfaces.

Results

After 2 days, we observed approximately 10⁴ CFU/ml biofilm-bound S. epidermidis (10³ CFU/ml initial population) on the antibiotics-loaded bone cement samples in the presence of hOB, while no bacteria were detected without hOB. No biofilm-bound bacteria were detectable after 7 days in either case. Similar levels of planktonic bacteria were observed on day 2 with and without hOB. After 7 days, about 10⁵ CFU/ml planktonic bacteria were present, but only in the absence of hOB. Further, no bacteria were observed within the biofilm, while the number of hOB was decreased to 10% of its initial value compared to 150% in the mono-culture of hOB.

Conclusion

We developed a co-culture setup that serves as a more comprehensive in vitro model for the onset of implant-associated infections and provides a test method for antimicrobial implant materials and coatings. We demonstrate that observations can be made that are unavailable from mono-culture experiments.     

Injection of hydrogen gas stimulates acid mine drainage treatment in laboratory‐scale hydroponic root mats

The environmentally benign disposal of acid mine drainage (AMD) is still a technical challenge. In the present study, artificial AMD was treated in a laboratory‐scale floating hydroponic root mat of soft rush, Juncus effusus. This ecotechnological system was operated with hydrogen injection and water recirculation but without an external carbon supply. It achieved a mean increase of ΔpH = 3.3 up to pH ≈ 8.2, high sulfate removal of up to 87%, and efficient removal of iron (100%), aluminum (99.8%), manganese (97.4%), and zinc (99.6%). Sulfide was not detected in the outflow. Treatment performance correlated with the amount of hydrogen loading. Daily oscillations of the redox potential up to amplitudes of ΔE_h ≈ 450 mV in a mean range of E_h ≈ ?150 to +300 mV indicated a correlation of plant physiology and removal processes. Apparently, sulfate and metal removal were the result of chemolithotrophic microbial sulfate reduction supported by the externally provided H₂ and chemoorganotrophic sulfate reduction driven by rhizodeposits. Bicarbonate generated in the microbial transformation of such plant‐derived organic carbon contributed to pH neutralization. The effluent's pH increase was governed further by recirculation of the treated AMD. The flow regime and the injection of hydrogen at the ground of the root mat caused concentration gradients where the most efficient removal occurred in the deepest zone of the root mat. Further investigations should target long‐term stability, plant growth dynamics, load variations, balances of carbon and sulfur, the removal of H₂S and metal precipitates from the system as well as efficient hydrogen supply.     

Insights into effective RNAi gained from large-scale siRNA validation screening

Transfection of chemically synthesized short interfering RNAs (siRNAs) enables a high level of sequence-specific gene silencing. Although siRNA design algorithms have been improved in recent years, it is still necessary to prove the functionality of a given siRNA experimentally. We have functionally tested several thousand siRNAs for target genes from various gene families including kinases, phosphatases, and cancer-related genes (e.g., genes involved in apoptosis and the cell cycle). Some targets were difficult to silence above a threshold of 70% knockdown. By working with one design algorithm and a standardized validation procedure, we discovered that the level of silencing achieved was not exclusively dependent on the siRNA sequences. Here we present data showing that neither the gene expression level nor the cellular environment has a direct impact on the knockdown which can be achieved for a given target. Modifications of the experimental setting have been investigated with the aim of improving knockdown efficiencies for siRNA-target combinations that show only moderate knockdown. Use of higher siRNA concentrations did not change the overall performance of the siRNA-target combinations analyzed. Optimal knockdown at the mRNA level was usually reached 48-72 hours after transfection. Target gene-specific characteristics such as the accessibility of the corresponding target sequences to the RNAi machinery appear to have a significant influence on the knockdown observed, making certain targets easy or difficult to knock down using siRNA.