Immunohistochemistry in the evaluation of neovascularization in tumor xenografts

Angiogenesis, or neovascularization, is known to play an important role in the neoplastic progression leading to metastasis. CD31 or Factor VIII-related antigen (F VIII RAg) immunohistochemistry is widely used in experimental studies for quantifying tumor neovascularization in immunocompromised animal models implanted with transformed human cell lines. Quantification, however, can be affected by variations in the methodology used to measure vascularization including antibody selection, antigen retrieval (AR) pretreatment, and evaluation techniques. To examine this further, we investigated the microvessel density (MVD) and the intensity of microvascular staining among five different human tumor xenografts and a mouse syngeneic tumor using anti-CD31 and F VIII RAg immunohistochemical staining. Different AR methods also were evaluated. Maximal retrieval of CD31 was achieved using 0.5 M Tris (pH 10) buffer, while maximum retrieval of F VIII RAg was achieved using 0.05% pepsin treatment of tissue sections. For each optimized retrieval condition, anti-CD31 highlighted small vessels better than F VIII RAg. Furthermore, the MVD of CD31 was significantly greater than that of F VIII RAg decorated vessels (p<0.001). The choice of antibody and AR method has a significant affect on immunohistochemical findings when studying angiogenesis. One also must use caution when comparing studies in the literature that use different techniques and reagents.     

The C. elegans Male Exercises Directional Control during Mating through Cholinergic Regulation of Sex-Shared Command Interneurons

Background

Mating behaviors in simple invertebrate model organisms represent tractable paradigms for understanding the neural bases of sex-specific behaviors, decision-making and sensorimotor integration. However, there are few examples where such neural circuits have been defined at high resolution or interrogated.

Methodology/Principal Findings

Here we exploit the simplicity of the nematode Caenorhabditis elegans to define the neural circuits underlying the male’s decision to initiate mating in response to contact with a mate. Mate contact is sensed by male-specific sensilla of the tail, the rays, which subsequently induce and guide a contact-based search of the hermaphrodite’s surface for the vulva (the vulva search). Atypically, search locomotion has a backward directional bias so its implementation requires overcoming an intrinsic bias for forward movement, set by activity of the sex-shared locomotory system. Using optogenetics, cell-specific ablation- and mutant behavioral analyses, we show that the male makes this shift by manipulating the activity of command cells within this sex-shared locomotory system. The rays control the command interneurons through the male-specific, decision-making interneuron PVY and its auxiliary cell PVX. Unlike many sex-shared pathways, PVY/PVX regulate the command cells via cholinergic, rather than glutamatergic transmission, a feature that likely contributes to response specificity and coordinates directional movement with other cholinergic-dependent motor behaviors of the mating sequence. PVY/PVX preferentially activate the backward, and not forward, command cells because of a bias in synaptic inputs and the distribution of key cholinergic receptors (encoded by the genes acr-18, acr-16 and unc-29) in favor of the backward command cells.

Conclusion/Significance

Our interrogation of male neural circuits reveals that a sex-specific response to the opposite sex is conferred by a male-specific pathway that renders subordinate, sex-shared motor programs responsive to mate cues. Circuit modifications of these types may make prominent contributions to natural variations in behavior that ultimately bring about speciation.     

Thermostable designed ankyrin repeat proteins (DARPins) as building blocks for innovative drugs

Designed ankyrin repeat proteins (DARPins) are antibody mimetics with high and mostly unexplored potential in drug development. By using in silico analysis and a rationally guided Ala scanning, we identified position 17 of the N-terminal capping repeat to play a key role in overall protein thermostability. The melting temperature of a DARPin domain with a single full-consensus internal repeat was increased by 8 °C to 10 °C when Asp17 was replaced by Leu, Val, Ile, Met, Ala, or Thr. We then transferred the Asp17Leu mutation to various backgrounds, including clinically validated DARPin domains, such as the vascular endothelial growth factor-binding domain of the DARPin abicipar pegol. In all cases, these proteins showed improvements in the thermostability on the order of 8 °C to 16 °C, suggesting the replacement of Asp17 could be generically applicable to this drug class. Molecular dynamics simulations showed that the Asp17Leu mutation reduces electrostatic repulsion and improves van-der-Waals packing, rendering the DARPin domain less flexible and more stable. Interestingly, this beneficial Asp17Leu mutation is present in the N-terminal caps of three of the five DARPin domains of ensovibep, a SARS-CoV-2 entry inhibitor currently in clinical development, indicating this mutation could be partly responsible for the very high melting temperature (>90 °C) of this promising anti-COVID-19 drug. Overall, such N-terminal capping repeats with increased thermostability seem to be beneficial for the development of innovative drugs based on DARPins.     

Role of native topology investigated by multiple unfolding simulations of four SH3 domains

The relative importance of amino acid sequence and native topology in the unfolding process of two SH3 domains and two circular permutants was investigated by 120 molecular dynamics runs at 375 K for a total simulation time of 0.72 micros. The alpha-spectrin (aSH3) and src SH3 (sSH3) domains, which have the same topology and a sequence identity of only 34%, show similar unfolding pathways. The disappearance of the three-stranded antiparallel beta-sheet is the last unfolding event, in agreement with a large repertoire of kinetic data derived from point mutations as well as glycine insertions and disulfide crosslinks. Two alternative routes of beta-sheet unfolding have emerged from the analysis of the trajectories. One is statistically preferred in aSH3 (n-src loop breaks before distal hair-pin) and the inverse in sSH3. An elongation of the beta2-beta3 hairpin was observed during the unfolding of sSH3 at 375 K and in 300 K simulations started from the putative transition state of sSH3 in accord with unusual kinetic data for point mutations at the n-src loop. The change of connectivity in the permutants influenced the sequence of unfolding events mainly at the permutation site. Regions where the connectivity remained unaffected showed the same chronology of contact disappearance. Taken together with previous folding simulations of two designed three-stranded antiparallel beta-sheet peptides, these results indicate that, at least for small beta-sheet proteins, the folding mechanism is primarily defined by the native state topology, whilst specific interactions determine the statistically predominant folding route.     

The role of flexibility and hydration on the sequence-specific DNA recognition by the Tn916 integrase protein: a molecular dynamics analysis

The N-terminal domain of the Tn916 integrase protein (INT-DBD) is responsible for DNA binding in the process of strand cleavage and joining reactions required for transposition of the Tn916 conjugative transposon. Site-specific association is facilitated by numerous protein-DNA contacts from the face of a three-stranded beta-sheet inserted into the major groove. The protein undergoes a subtle conformational transition and is slightly unfolded in the protein-DNA complex. The conformation of many charged residues is poorly defined by NMR data but mutational studies have indicated that removal of polar side chains decreases binding affinity, while non-polar contacts are malleable. Based on analysis of the binding enthalpy and binding heat capacity, we have reasoned that dehydration of the protein-DNA interface is incomplete. This study presents results from a molecular dynamics investigation of the INT-DBD-DNA complex aimed at a more detailed understanding of the role of conformational dynamics and hydration in site-specific binding. Comparison of simulations (total of 13 ns) of the free protein and of the bound protein conformation (in isolation or DNA-bound) reveals intrinsic flexibility in certain parts of the molecule. Conformational adaptation linked to partial unfolding appears to be induced by protein-DNA contacts. The protein-DNA hydrogen-bonding network is highly dynamic. The simulation identifies protein-DNA interactions that are poorly resolved or only surmised from the NMR ensemble. Single water molecules and water clusters dynamically optimize the complementarity of polar interactions at the 'wet' protein-DNA interface. The simulation results are useful to establish a qualitative link between experimental data on individual residue's contribution to binding affinity and thermodynamic properties of INT-DBD alone and in complex with DNA.     

Dietary input of microbes and host genetic variation shape among-population differences in stickleback gut microbiota

To explain differences in gut microbial communities we must determine how processes regulating microbial community assembly (colonization, persistence) differ among hosts and affect microbiota composition. We surveyed the gut microbiota of threespine stickleback (Gasterosteus aculeatus) from 10 geographically clustered populations and sequenced environmental samples to track potential colonizing microbes and quantify the effects of host environment and genotype. Gut microbiota composition and diversity varied among populations. These among-population differences were associated with multiple covarying ecological variables: habitat type (lake, stream, estuary), lake geomorphology and food- (but not water-) associated microbiota. Fish genotype also covaried with gut microbiota composition; more genetically divergent populations exhibited more divergent gut microbiota. Our results suggest that population level differences in stickleback gut microbiota may depend more on internal sorting processes (host genotype) than on colonization processes (transient environmental effects).     

High-Dose Benzodiazepine Dependence: A Qualitative Study of Patients’ Perceptions on Initiation,Reasons for Use,and Obtainment

Background

High-dose benzodiazepine (BZD) dependence is associated with a wide variety of negative health consequences. Affected individuals are reported to suffer from severe mental disorders and are often unable to achieve long-term abstinence via recommended discontinuation strategies. Although it is increasingly understood that treatment interventions should take subjective experiences and beliefs into account, the perceptions of this group of individuals remain under-investigated.

Methods

We conducted an exploratory qualitative study with 41 adult subjects meeting criteria for (high-dose) BZD-dependence, as defined by ICD-10. One-on-one in-depth interviews allowed for an exploration of this group’s views on the reasons behind their initial and then continued use of BZDs, as well as their procurement strategies. Mayring’s qualitative content analysis was used to evaluate our data.

Results

In this sample, all participants had developed explanatory models for why they began using BZDs. We identified a multitude of reasons that we grouped into four broad categories, as explaining continued BZD use: (1) to cope with symptoms of psychological distress or mental disorder other than substance use, (2) to manage symptoms of physical or psychological discomfort associated with somatic disorder, (3) to alleviate symptoms of substance-related disorders, and (4) for recreational purposes, that is, sensation-seeking and other social reasons. Subjects often considered BZDs less dangerous than other substances and associated their use more often with harm reduction than as recreational. Specific obtainment strategies varied widely: the majority of participants oscillated between legal and illegal methods, often relying on the black market when faced with treatment termination.

Conclusions

Irrespective of comorbidity, participants expressed a clear preference for medically related explanatory models for their BZD use. We therefore suggest that clinicians consider patients’ motives for long-term, high-dose BZD use when formulating treatment plans for this patient group, especially since it is known that individuals are more compliant with approaches they perceive to be manageable, tolerable, and effective.     

Caspase-mediated cleavage of the exosome subunit PM/Scl-75 during apoptosis

Recent studies have implicated the dying cell as a potential reservoir of modified autoantigens that might initiate and drive systemic autoimmunity in susceptible hosts. A number of subunits of the exosome, a complex of 3'→5' exoribonucleases that functions in a variety of cellular processes, are recognized by the so-called anti-PM/Scl autoantibodies, found predominantly in patients suffering from an overlap syndrome of myositis and scleroderma. Here we show that one of these subunits, PM/Scl-75, is cleaved during apoptosis. PM/Scl-75 cleavage is inhibited by several different caspase inhibitors. The analysis of PM/Scl-75 cleavage by recombinant caspase proteins shows that PM/Scl-75 is efficiently cleaved by caspase-1, to a smaller extent by caspase-8, and relatively inefficiently by caspase-3 and caspase-7. Cleavage of the PM/Scl-75 protein occurs in the C-terminal part of the protein at Asp369 (IILD³⁶⁹↓G), and at least a fraction of the resulting N-terminal fragments of PM/Scl-75 remains associated with the exosome. Finally, the implications of PM/Scl-75 cleavage for exosome function and the generation of anti-PM/Scl-75 autoantibodies are discussed.     

Functional plasticity in the substrate binding site of beta-secretase

The aspartic protease beta-secretase (BACE) cleaves the amyloid precursor protein into a 42 residue beta-peptide, which is the principal biochemical marker of Alzheimer's disease. Multiple explicit-water molecular dynamics simulations of the apo and inhibitor bound structures of BACE indicate that both open- and closed-flap conformations are accessible at room temperature and should be taken into account for inhibitor design. Correlated motion is observed within each of the two lobes of BACE, as well as for the interfacial region. A self-inhibited conformation with the side chain of Tyr71 occupying the S(1) pocket is present in some of the unbound simulations. The reversible loss of the side chain hydrogen bond between the catalytic Asp32 and Ser35, due to the concomitant reorientation of the Ser35 hydroxyl group and a water molecule conserved in pepsin-like enzymes, provides further evidence for the suggestion that Ser35 assists in proton acceptance and release by Asp32 during catalysis.