Low frequency vibrations of amino acid homopolymers observed by synchrotron far‐ir absorption spectroscopy: Excited state effects dominate the temperature dependence of the spectra

Synchrotron ir radiation was used to collect far‐ir spectroscopy data on amino acid homopolymers, including poly‐L ‐phenylalanine, poly‐L ‐alanine, poly‐L ‐leucine, poly‐L ‐valine, and poly‐L ‐tryptophan. The intensity of the broadband synchrotron ir source, coupled with the use of a Michelson interferometer and Fourier transformed data, provides uniquely high signal to noise far‐ir data, which can be analyzed by deconvolution techniques that are well established. The temperature dependence of the spectra reveals consistent trends, with the bands shifting to higher frequencies and intensities as the temperature is lowered. A careful analysis of poly‐L ‐phenylalanine, as a function of temperature from 10 to 295 K, reveals consistent (monotonic) changes with temperature. An explanation for these effects is presented. Since the observed modes are of low frequency and low energy, a substantial fraction of the observed transitions result from a combination of excited state absorption and stimulated emission at ambient temperature. Anharmonicity of the vibrational potentials, with the resultant decrease in spacing of higher energy levels, is consistent with the frequency up shifts as temperature is decreased. The results emphasize that these excited state effects likely govern much of the behavior of low‐frequency modes in all biomolecules. © 1999 John Wiley & Sons, Inc. Biopoly 49: 591–603, 1999     

Decline of unique Pontocaspian biodiversity in the Black Sea Basin: A review

The unique aquatic Pontocaspian (PC) biota of the Black Sea Basin (BSB) is in decline. The lack of detailed knowledge on the status and trends of species, populations, and communities hampers a thorough risk assessment and precludes effective conservation. This paper reviews PC biodiversity trends in the BSB (Bulgaria, Romania, Moldova, Ukraine, and Russia) using endemic mollusks as a model group. We aim to assess changes in PC habitats, community structure, and species distribution over the past century and to identify direct anthropogenic threats. The presence/absence data of target mollusk species were assembled from literature, reports, and personal observations. Pontocaspian biodiversity trends in the northwestern BSB coastal regions were established by comparing 20th‐ and 21st‐century occurrences. The direct drivers of habitat and biodiversity change were identified and documented. We found that a pronounced decline of PC species and communities is driven by (a) damming of rivers, (b) habitat modifications that disturbed previous natural salinity gradients and settings in the studied area, (c) pollution and eutrophication, (d) invasive alien species, and (e) climate change. Four out of the 10 studied regions, namely, the Danube Delta–Razim Lake system, Dniester Liman, Dnieper–Bug estuary, and Taganrog Bay–Don Delta, contain favorable ecological conditions for PC communities and still host threatened endemic PC mollusk species. Distribution data are incomplete, but the scale of deterioration of PC species and communities is evident from the assembled data, as are major direct threats. Pontocaspian biodiversity in the BSB is profoundly affected by human activities. Standardized observation and collection data as well as precise definition of PC biota and habitats are necessary for targeted conservation actions. This study will help to set the research and policy agenda required to improve data collection to accommodate effective conservation of the unique PC biota.     

CD47 associates with alpha 5 integrin and regulates responses of human articular chondrocytes to mechanical stimulation in an <Emphasis Type="Italic">in vitro</Emphasis>model

Background  

Recent studies provide evidence of roles for integrins in mechanical signalling in bone and cartilage. Integrin signalling is modulated by various mechanisms, including interaction with other transmembrane proteins. We aimed to identify whether one such protein, integrin-associated protein (CD47/IAP), is expressed by chondrocytes and whether it may regulate integrin-dependent mechanotransduction.     

The V1-V3 region of a brain-derived HIV-1 envelope glycoprotein determines macrophage tropism,low CD4 dependence,increased fusogenicity and altered sensitivity to entry inhibitors

Background

HIV-1 infects macrophages and microglia in the brain and can cause neurological disorders in infected patients. We and others have shown that brain-derived envelope glycoproteins (Env) have lower CD4 dependence and higher avidity for CD4 than those from peripheral isolates, and we have also observed increased fusogenicity and reduced sensitivity to the fusion inhibitor T-1249. Due to the genetic differences between brain and spleen env from one individual throughout gp120 and in gp41's heptad repeat 2 (HR2), we investigated the viral determinants for the phenotypic differences by performing functional studies with chimeric and mutant Env.

Results

Chimeric Env showed that the V1/V2-C2-V3 region in brain's gp120 determines the low CD4 dependence and high avidity for CD4, as well as macrophage tropism and reduced sensitivity to the small molecule BMS-378806. Changes in brain gp41's HR2 region did not contribute to the increased fusogenicity or to the reduced sensitivity to T-1249, since a T-1249-based peptide containing residues found in brain's but not in spleen's HR2 had similar potency than T-1249 and interacted similarly with an immobilized heptad repeat 1-derived peptide in surface plasmon resonance analysis. However, the increased fusogenicity and reduced T-1249 sensitivity of brain and certain chimeric Env mostly correlated with the low CD4 dependence and high avidity for CD4 determined by brain's V1-V3 region. Remarkably, most but not all of these low CD4-dependent, macrophage tropic envelopes glycoproteins also had increased sensitivity to the novel allosteric entry inhibitor HNG-105. The gp120's C2 region asparagine 283 (N283) has been previously associated with macrophage tropism, brain infection, lower CD4 dependence and higher CD4 affinity. Therefore, we introduced the N283T mutation into an env clone from a brain-derived isolate and into a brain tissue-derived env clone, and the T283N change into a spleen-derived env from the same individual; however, we found that their phenotypes were not affected.

Conclusion

We have identified that the V1-V3 region of a brain-derived envelope glycoprotein seems to play a crucial role in determining not only the low CD4 dependence and increased macrophage tropism, but also the augmented fusogenicity and reduced sensitivity to T-1249 and BMS-378806. By contrast, increased sensitivity to HNG-105 mostly correlated with low CD4 dependence and macrophage tropism but was not determined by the presence of the brain's V1-V3 region, confirming that viral determinants of phenotypic changes in brain-derived envelope glycoproteins are likely complex and context-dependent.

     

Single cell-derived spheroids capture the self-renewing subpopulations of metastatic ovarian cancer

High Grade Serous Ovarian cancer (HGSOC) is a major unmet need in oncology, due to its precocious dissemination and the lack of meaningful human models for the investigation of disease pathogenesis in a patient-specific manner. To overcome this roadblock, we present a new method to isolate and grow single cells directly from patients’ metastatic ascites, establishing the conditions for propagating them as 3D cultures that we refer to as single cell-derived metastatic ovarian cancer spheroids (sMOCS). By single cell RNA sequencing (scRNAseq) we define the cellular composition of metastatic ascites and trace its propagation in 2D and 3D culture paradigms, finding that sMOCS retain and amplify key subpopulations from the original patients’ samples and recapitulate features of the original metastasis that do not emerge from classical 2D culture, including retention of individual patients’ specificities. By enabling the enrichment of uniquely informative cell subpopulations from HGSOC metastasis and the clonal interrogation of their diversity at the functional and molecular level, this method provides a powerful instrument for precision oncology in ovarian cancer.Subject terms: Cancer models, Cancer stem cells, Experimental models of disease     

Axial Dynamics, Stability, and Interspecies Similarity of Bacterial Community Structure in the Highly Compartmentalized Gut of Soil-Feeding Termites (Cubitermes spp.)

The highly compartmentalized gut of soil-feeding termites is characterized by pronounced axial dynamics in physicochemical conditions and microbial processes. In a companion paper (D. Schmitt-Wagner, M. W. Friedrich, B. Wagner, and A. Brune, Appl. Environ. Microbiol. 69:6007-6017, 2003), we demonstrated that the variety of physicochemical conditions in the different gut compartments of Cubitermes spp. is reflected in the diversity of the respective intestinal microbial communities. Here, we used molecular fingerprints of 16S rRNA genes of the bacterial community, obtained by terminal restriction fragment length polymorphism (T-RFLP) analysis, to describe the axial dynamics of the bacterial community structure in the different gut sections. Comparison of the T-RFLP profiles with the predicted terminal restriction fragments of the clones in clone libraries of the gut segments in Cubitermes orthognathus confirmed that all hindgut sections harbored distinct bacterial communities. Morisita indices of community similarity, calculated by comparing the different patterns, revealed large differences between the bacterial communities of soil, gut, and nest material and also among the individual gut sections. By contrast, comparison of the homologous gut segments of different Cubitermes species indicated that the three termite species investigated possessed a similar, gut-specific microbiota that remained comparatively stable even during several months of maintenance in the laboratory.     

Development of a Universal Microarray Based on the Ligation Detection Reaction and 16S rRNA Gene Polymorphism To Target Diversity of Cyanobacteria

The cyanobacteria are photosynthetic prokaryotes of significant ecological and biotechnological interest, since they strongly contribute to primary production and are a rich source of bioactive compounds. In eutrophic fresh and brackish waters, their mass occurrences (water blooms) are often toxic and constitute a high potential risk for human health. Therefore, rapid and reliable identification of cyanobacterial species in complex environmental samples is important. Here we describe the development and validation of a microarray for the identification of cyanobacteria in aquatic environments. Our approach is based on the use of a ligation detection reaction coupled to a universal array. Probes were designed for detecting 19 cyanobacterial groups including Anabaena/Aphanizomenon, Calothrix, Cylindrospermopsis, Cylindrospermum, Gloeothece, halotolerants, Leptolyngbya, Palau Lyngbya, Microcystis, Nodularia, Nostoc, Planktothrix, Antarctic Phormidium, Prochlorococcus, Spirulina, Synechococcus, Synechocystis, Trichodesmium, and Woronichinia. These groups were identified based on an alignment of over 300 cyanobacterial 16S rRNA sequences. For validation of the microarrays, 95 samples (24 axenic strains from culture collections, 27 isolated strains, and 44 cloned fragments recovered from environmental samples) were tested. The results demonstrated a high discriminative power and sensitivity to 1 fmol of the PCR-amplified 16S rRNA gene. Accurate identification of target strains was also achieved with unbalanced mixes of PCR amplicons from different cyanobacteria and an environmental sample. Our universal array method shows great potential for rapid and reliable identification of cyanobacteria. It can be easily adapted to future development and could thus be applied both in research and environmental monitoring.     

Sequential anaerobic–aerobic degradation of munitions waste

A sequential anaerobic–aerobic biodegradation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) was studied. The results demonstrated that: (i) a complete degradation of RDX was achieved within 20 days using a consortium of bacteria from a wastewater activated sludge, (ii) RDX degradation did not occur under aerobic conditions alone, (iii) RDX-degrading bacterial strain that was isolated from the activated sludge completely degraded RDX within 2 days, and (iv) RDX- induced protein expressions were observed in the RDX-degrading bacterial strain. Based on fatty acid composition and a confirmation with a 16S rRNA analysis, the RDX-degrading bacterial strain was identified as a Bacillus pumilus—GC subgroup B.