Retinal proteome profiling of inherited retinal degeneration across three different mouse models suggests common drug targets in retinitis pigmentosa.

Inherited retinal degenerations (IRDs) are a leading cause of blindness among the population of young people in the developed world. Approximately half of IRDs initially manifest as gradual loss of night vision and visual fields, characteristic of retinitis pigmentosa (RP). Due to challenges in genetic testing, and the large heterogeneity of mutations underlying RP, targeted gene therapies are an impractical largescale solution in the foreseeable future. For this reason, identifying key pathophysiological pathways in IRDs that could be targets for mutation-agnostic and disease-modifying therapies (DMTs) is warranted. In this study, we investigated the retinal proteome of three distinct IRD mouse models, in comparison to sex- and age-matched wild-type mice. Specifically, we used the Pde6ßRd10 (rd10) and RhoP23H/WT (P23H) mouse models of autosomal recessive and autosomal dominant RP, respectively, as well as the Rpe65-/- mouse model of Leber´s congenital amaurosis type 2 (LCA2). The mice were housed at two distinct institutions and analyzed using LC-MS in three separate facilities/instruments following data-dependent and data-independent acquisition modes. This cross-institutional and multi-methodological approach signifies the reliability and reproducibility of the results. The largescale profiling of the retinal proteome, coupled with in vivo electroretinography recordings, provided us with a reliable basis for comparing the disease phenotypes and severity. Despite evident inflammation, cellular stress, and downscaled phototransduction observed consistently across all three models, the underlying pathologies of RP and LCA2 displayed many differences, sharing only four general KEGG pathways. The opposite is true for the two RP models in which we identify remarkable convergence in proteomic phenotype even though the mechanism of primary rod death in rd10 and P23H mice is different. Our data highlights the cAMP and cGMP second-messenger signaling pathways as potential targets for therapeutic intervention. The proteomic data is curated and made publicly available, facilitating the discovery of universal therapeutic targets for RP.

Registered report: A pilot investigation of acute exercise response among girls and young women with and without eating disorders.

OBJECTIVE: Driven exercise (DEx) is a serious and common feature of eating disorders (EDs), but current understanding of factors that give rise to and maintain DEx is limited. DEx may be reinforced through its effects on the threat reduction and reward systems. The current protocol is designed to evaluate acute psychobiological response to exercise among female participants (age 16-22) with and without EDs. METHOD: Twenty medically-stable participants with restrictive-spectrum EDs and 20 healthy control (HC) participants will complete study screening and three task visits which will include two 30-minute bouts of aerobic exercise. RESULTS: We aim to validate and demonstrate feasibility of two tasks capturing exercise response in this sample. Further, we will estimate the degree to which a bout of exercise impacts state body image, affect, and circulating concentrations of biological markers among participants, and we will examine whether the impact of exercise on psychological outcomes may differ across ED and HC groups. DISCUSSION: Completion of this project will contribute to the conceptualization of DEx and how individuals' acute biological and affective responses to exercise contribute to risk for and maintenance of DEx.

Reframing mental illness: The role of essentialism on perceived treatment efficacy and stigmatization.

People believe that treatments for illnesses are effective when they target the cause of the illness. Prior work suggests that biological essentialist explanations of mental illness lead people to prefer medications or other pharmacological treatments. However, prior work has not distinguished between biological and essentialist explanations. In three studies (total n = 517), we presented adults with vignettes about an individual with an artificial mental illness and manipulated the descriptions to emphasize or de-emphasize essentialist characteristics. Critically, none of the vignettes made reference to a biological basis for the disorder. Participants rated their willingness to interact with the person described in the vignettes and how effective they believed drug treatment and talk therapy would be on the mental illness. Across the three studies, describing mental illness with an essentialist framing led participants to think drug treatments would be more effective, but there was no effect for stigma or perceived effectiveness of talk therapy. This effect appears to be mediated by how much participants essentialized individuals with the disorder. The first framing that participants encountered seemed to shape their reasoning for the remainder of the study, even if they saw conflicting framing later on. The framing manipulation had similar effects for individuals with and without a mental illness. Results suggest that it is important to consider how mental illness is framed to the general public as it might impact people's treatment preferences.

Metaproteomic and Metabolomic Approaches for Characterizing the Gut Microbiome.

The gut microbiome has been shown to play a significant role in human healthy and diseased states. The dynamic signaling that occurs between the host and microbiome is critical for the maintenance of host homeostasis. Analyzing the human microbiome with metaproteomics, metabolomics, and integrative multi-omics analyses can provide significant information on markers for healthy and diseased states, allowing for the eventual creation of microbiome-targeted treatments for diseases associated with dysbiosis. Metaproteomics enables functional activity information to be gained from the microbiome samples, while metabolomics provides insight into the overall metabolic states affecting/representing the host-microbiome interactions. Combining these functional -omic platforms together with microbiome composition profiling allows for a holistic overview on the functional and metabolic state of the microbiome and its influence on human health. Here the benefits of metaproteomics, metabolomics, and the integrative multi-omic approaches to investigating the gut microbiome in the context of human health and diseases are reviewed.

Novel Stenotrophomonas maltophilia temperate phage DLP4 is capable of lysogenic conversion.

BACKGROUND: Temperate bacteriophages are capable of lysogenic conversion of new bacterial hosts. This phenomenon is often ascribed to "moron" elements that are acquired horizontally and transcribed independently from the rest of the phage genes. Whereas some bacterial species exhibit relatively little prophage-dependent phenotypic changes, other bacterial species such as Stenotrophomonas maltophilia appear to commonly adopt prophage genetic contributions. RESULTS: The novel S. maltophilia bacteriophage DLP4 was isolated from soil using the highly antibiotic-resistant S. maltophilia strain D1585. Genome sequence analysis and functionality testing showed that DLP4 is a temperate phage capable of lysogenizing D1585. Two moron genes of interest, folA (BIT20_024) and ybiA (BIT20_065), were identified and investigated for their putative activities using complementation testing and phenotypic and transcriptomic changes between wild-type D1585 and the D1585::DLP4 lysogen. The gp24 / folA gene encodes dihydrofolate reductase (DHFR: FolA), an enzyme responsible for resistance to the antibiotic trimethoprim. I-TASSER analysis of DLP4 FolA predicted structural similarity to Bacillus anthracis DHFR and minimum inhibitory concentration experiments demonstrated that lysogenic conversion of D1585 by DLP4 provided the host cell with an increase in trimethoprim resistance. The gp65 / ybiA gene encodes N-glycosidase YbiA, which in E. coli BW25113 is required for its swarming motility phenotype. Expressing DLP4 ybiA in strain ybiA770(del)::kan restored its swarming motility activity to wildtype levels. Reverse transcription-PCR confirmed the expression of both of these genes during DLP4 lysogeny. CONCLUSIONS: S. maltophilia temperate phage DLP4 contributes to the antibiotic resistance exhibited by its lysogenized host strain. Genomic analyses can greatly assist in the identification of phage moron genes potentially involved in lysogenic conversion. Further research is required to fully understand the specific contributions temperate phage moron genes provide with respect to the antibiotic resistance and virulence of S. maltophilia host cells.

The isolation and characterization of two Stenotrophomonas maltophilia bacteriophages capable of cross-taxonomic order infectivity.

BACKGROUND: A rapid worldwide increase in the number of human infections caused by the extremely antibiotic resistant bacterium Stenotrophomonas maltophilia is prompting alarm. One potential treatment solution to the current antibiotic resistance dilemma is "phage therapy", the clinical application of bacteriophages to selectively kill bacteria. RESULTS: Towards that end, phages DLP1 and DLP2 (vB_SmaS-DLP_1 and vB_SmaS-DLP_2, respectively) were isolated against S. maltophilia strain D1585. Host range analysis for each phage was conducted using 27 clinical S. maltophilia isolates and 11 Pseudomonas aeruginosa strains. Both phages exhibit unusually broad host ranges capable of infecting bacteria across taxonomic orders. Transmission electron microscopy of the phage DLP1 and DLP2 morphology reveals that they belong to the Siphoviridae family of bacteriophages. Restriction fragment length polymorphism analysis and complete genome sequencing and analysis indicates that phages DLP1 and DLP2 are closely related but different phages, sharing 96.7 % identity over 97.2 % of their genomes. These two phages are also related to P. aeruginosa phages vB_Pae-Kakheti_25 (PA25), PA73, and vB_PaeS_SCH_Ab26 (Ab26) and more distantly related to Burkholderia cepacia complex phage KL1, which together make up a taxonomic sub-family. Phages DLP1 and DLP2 exhibited significant differences in host ranges and growth kinetics. CONCLUSIONS: The isolation and characterization of phages able to infect two completely different species of bacteria is an exciting discovery, as phages typically can only infect related bacterial species, and rarely infect bacteria across taxonomic families, let alone across taxonomic orders.

Functional domains of Acinetobacter bacteriophage tail fibers.

A rapid increase in antimicrobial resistant bacterial infections around the world is causing a global health crisis. The Gram-negative bacterium Acinetobacter baumannii is categorized as a Priority 1 pathogen for research and development of new antimicrobials by the World Health Organization due to its numerous intrinsic antibiotic resistance mechanisms and ability to quickly acquire new resistance determinants. Specialized phage enzymes, called depolymerases, degrade the bacterial capsule polysaccharide layer and show therapeutic potential by sensitizing the bacterium to phages, select antibiotics, and serum killing. The functional domains responsible for the capsule degradation activity are often found in the tail fibers of select A. baumannii phages. To further explore the functional domains associated with depolymerase activity, tail-associated proteins of 71 sequenced and fully characterized phages were identified from published literature and analyzed for functional domains using InterProScan. Multisequence alignments and phylogenetic analyses were conducted on the domain groups and assessed in the context of noted halo formation or depolymerase characterization. Proteins derived from phages noted to have halo formation or a functional depolymerase, but no functional domain hits, were modeled with AlphaFold2 Multimer, and compared to other protein models using the DALI server. The domains associated with depolymerase function were pectin lyase-like (SSF51126), tailspike binding (cd20481), (Trans)glycosidases (SSF51445), and potentially SGNH hydrolases. These findings expand our knowledge on phage depolymerases, enabling researchers to better exploit these enzymes for therapeutic use in combating the antimicrobial resistance crisis.

Characterization of Virulent T4-Like Acinetobacter baumannii Bacteriophages DLP1 and DLP2.

The world is currently facing a global health crisis due to the rapid increase in antimicrobial-resistant bacterial infections. One of the most concerning pathogens is Acinetobacter baumannii, which is listed as a Priority 1 pathogen by the World Health Organization. This Gram-negative bacterium has many intrinsic antibiotic resistance mechanisms and the ability to quickly acquire new resistance determinants from its environment. A limited number of effective antibiotics against this pathogen complicates the treatment of A. baumannii infections. A potential treatment option that is rapidly gaining interest is "phage therapy", or the clinical application of bacteriophages to selectively kill bacteria. The myoviruses DLP1 and DLP2 (vB_AbaM-DLP_1 and vB_AbaM-DLP_2, respectively) were isolated from sewage samples using a capsule minus variant of A. baumannii strain AB5075. Host range analysis of these phages against 107 A. baumannii strains shows a limited host range, infecting 15 and 21 for phages DLP1 and DLP2, respectively. Phage DLP1 has a large burst size of 239 PFU/cell, a latency period of 20 min, and virulence index of 0.93. In contrast, DLP2 has a smaller burst size of 24 PFU/cell, a latency period of 20 min, and virulence index of 0.86. Both phages show potential for use as therapeutics to combat A. baumannii infections.

Combining bacteriophage and vancomycin is efficacious against MRSA biofilm-like aggregates formed in synovial fluid.

Background: Biofilm formation is a major clinical challenge contributing to treatment failure of periprosthetic joint infection (PJI). Lytic bacteriophages (phages) can target biofilm associated bacteria at localized sites of infection. The aim of this study is to investigate whether combination therapy of phage and vancomycin is capable of clearing Staphylococcus aureus biofilm-like aggregates formed in human synovial fluid. Methods: In this study, S. aureus BP043, a PJI clinical isolate was utilized. This strain is a methicillin-resistant S. aureus (MRSA) biofilm-former. Phage Remus, known to infect S. aureus, was selected for the treatment protocol. BP043 was grown as aggregates in human synovial fluid. The characterization of S. aureus aggregates was assessed for structure and size using scanning electron microscopy (SEM) and flow cytometry, respectively. Moreover, the formed aggregates were subsequently treated in vitro with: (a) phage Remus [∼108 plaque-forming units (PFU)/ml], (b) vancomycin (500 µg/ml), or (c) phage Remus (∼108 PFU/ml) followed by vancomycin (500 µg/ml), for 48 h. Bacterial survival was quantified by enumeration [colony-forming units (CFU)/ml]. The efficacy of phage and vancomycin against BP043 aggregates was assessed in vivo as individual treatments and in combination. The in vivo model utilized Galleria mellonella larvae which were infected with BP043 aggregates pre-formed in synovial fluid. Results: Scanning electron microscopy (SEM) images and flow cytometry data demonstrated the ability of human synovial fluid to promote formation of S. aureus aggregates. Treatment with Remus resulted in significant reduction in viable S. aureus residing within the synovial fluid aggregates compared to the aggregates that did not receive Remus (p < 0.0001). Remus was more efficient in eliminating viable bacteria within the aggregates compared to vancomycin (p < 0.0001). Combination treatment of Remus followed by vancomycin was more efficacious in reducing bacterial load compared to using either Remus or vancomycin alone (p = 0.0023, p < 0.0001, respectively). When tested in vivo, this combination treatment also resulted in the highest survival rate (37%) 96 h post-treatment, compared to untreated larvae (3%; p < 0.0001). Conclusion: We demonstrate that combining phage Remus and vancomycin led to synergistic interaction against MRSA biofilm-like aggregates in vitro and in vivo.

Bacteriophage Isolation, Purification, and Characterization Techniques Against Ubiquitous Opportunistic Pathogens.

Healthcare-associated infection with "ESKAPE" pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) is a global health crisis due to their extensive intrinsic antibiotic resistance and the ability to quickly acquire resistance determinants. Alternative treatment options are required to combat this crisis, and one possibility is the use of bacteriophages, or viruses that strictly infect the pathogenic bacteria. Currently, there is a renaissance in research and development into the use of phages to target multi-, extensively, and pan-resistant bacterial infections in humans, known as phage therapy. Using A. baumannii as an example, this article describes the isolation and purification of bacteriophages from sewage and soil samples, as well as general methods used in phage research such as precipitation of phages using polyethylene glycol, host range analysis, single-cell burst size determination, DNA extraction, and restriction fragment length polymorphism analysis. © 2022 National Research Council Canada. Current Protocols © 2022 Wiley Periodicals LLC. Reproduced with the permission of the Minister of Innovation, Science, and Industry. Basic Protocol 1: Isolation of bacteriophages against A. baumannii from sewage samples Alternate Protocol 1: Isolation of bacteriophages against A. baumannii from soil samples Support Protocol 1: Titering a bacteriophage stock Basic Protocol 2: Purification of phage to an axenic working stock Support Protocol 2: Liquid propagation of bacteriophage Basic Protocol 3: Host range analysis using the spot plate method Basic Protocol 4: Single burst size analysis Alternate Protocol 2: One-step growth curve Basic Protocol 5: Precipitation of bacteriophage using PEG 6000 Basic Protocol 6: DNA extraction from dsDNA bacteriophages Basic Protocol 7: Restriction fragment length polymorphism analysis of novel phage genomes.

Characterization of Novel Broad-Host-Range Bacteriophage DLP3 Specific to Stenotrophomonas maltophilia as a Potential Therapeutic Agent.

A novel Siphoviridae phage specific to the bacterial species Stenotrophomonas maltophilia was isolated from a pristine soil sample and characterized as a second member of the newly established Delepquintavirus genus. Phage DLP3 possesses one of the broadest host ranges of any S. maltophilia phage yet characterized, infecting 22 of 29 S. maltophilia strains. DLP3 has a genome size of 96,852 bp and a G+C content of 58.4%, which is significantly lower than S. maltophilia host strain D1571 (G+C content of 66.9%). The DLP3 genome encodes 153 coding domain sequences covering 95% of the genome, including five tRNA genes with different specificities. The DLP3 lysogen exhibits a growth rate increase during the exponential phase of growth as compared to the wild type strain. DLP3 also encodes a functional erythromycin resistance protein, causing lysogenic conversion of the host D1571 strain. Although a temperate phage, DLP3 demonstrates excellent therapeutic potential because it exhibits a broad host range, infects host cells through the S. maltophilia type IV pilus, and exhibits lytic activity in vivo. Undesirable traits, such as its temperate lifecycle, can be eliminated using genetic techniques to produce a modified phage useful in the treatment of S. maltophilia bacterial infections.

Fenestrated endovascular aneurysm repair with concomitant horseshoe kidney.

Horseshoe kidney (HSK) is a rare anatomic anomaly that poses surgical challenges in the setting of abdominal aortic aneurysm repair. An endovascular approach is safer for patients yet carries technical challenge because of variable renal vasculature. We present the case of a patient with an infrarenal abdominal aortic aneurysm and concomitant HSK who underwent successful repair with a custom fenestrated endograft with preservation of a midaortic renal artery. Complex endovascular aneurysm repair options for HSK include chimneys and fenestrated stent grafts. We suggest that the availability of custom fenestrated grafts and ongoing skill enhancement among vascular surgeons may make this approach more favorable.

Evaluation of Telepsychiatry-Enabled Perinatal Integrated Care.

OBJECTIVE: The purpose of this article is to describe the implementation of the first known telepsychiatry-enabled model of perinatal integrated care and to report initial results following implementation. METHODS: Behavioral health screening data were collected from 712 patients at an urban women's clinic, and a more in-depth set of process and outcome measures, including treatment engagement, services utilized, and delivery and postpartum patient outcomes, was collected from 135 patients referred for behavioral health services. Using nationally published metrics to provide context, the authors applied a descriptive design to evaluate and conduct analyses of program outcomes. RESULTS: The telehealth-enabled integrated care model was successfully implemented within a specialty obstetrics practice. Identification and treatment of behavioral health issues exceeded nationally published rates. The model was also associated with positive indices related to birth weight and breastfeeding behavior. CONCLUSIONS: These initial results point to telepsychiatry as an effective tool for expanding perinatal integrated care and lay the foundation for further study and model refinement. The results also add to the growing body of evidence for the use of telepsychiatry-supported integrated care across diverse clinical settings and patient populations.

Use of Greater Wax Moth Larvae (Galleria mellonella) as an Alternative Animal Infection Model for Analysis of Bacterial Pathogenesis.

Alternative infection models of bacterial pathogenesis are useful because they reproduce some of the disease characteristics observed in higher animals. Insect models are especially useful for modeling bacterial infections, as they are inexpensive, generally less labor-intensive, and more ethically acceptable than experimentation on higher organisms. Similar to animals, insects have been shown to possess innate immune systems that respond to pathogenic bacteria.

Complete Genome Sequence of Temperate Stenotrophomonas maltophilia Bacteriophage DLP5.

Stenotrophomonas maltophilia bacteriophage DLP5 is a temperate phage with Siphoviridae family morphotype. DLP5 (vB_SmaS_DLP_5) is the first S. maltophilia phage shown to exist as a phagemid. The DLP5 genome is 96,542 bp, encoding 149 open reading frames (ORFs), including four tRNAs. Genomic characterization reveals moron genes potentially involved in host cell membrane modification.

Identification and Characterization of Type IV Pili as the Cellular Receptor of Broad Host Range Stenotrophomonas maltophilia Bacteriophages DLP1 and DLP2.

Bacteriophages DLP1 and DLP2 are capable of infecting both Stenotrophomonas maltophilia and Pseudomonas aeruginosa strains, two highly antibiotic resistant bacterial pathogens, which is unusual for phages that typically exhibit extremely limited host range. To explain their unusual cross-order infectivity and differences in host range, we have identified the type IV pilus as the primary receptor for attachment. Screening of a P. aeruginosa PA01 mutant library, a host that is susceptible to DLP1 but not DLP2, identified DLP1-resistant mutants with disruptions in pilus structural and regulatory components. Subsequent complementation of the disrupted pilin subunit genes in PA01 restored DLP1 infection. Clean deletion of the major pilin subunit, pilA, in S. maltophilia strains D1585 and 280 prevented phage binding and lysis by both DLP1 and DLP2, and complementation restored infection by both. Transmission electron microscopy shows a clear interaction between DLP1 and pili of both D1585 and PA01. These results support the identity of the type IV pilus as the receptor for DLP1 and DLP2 infection across their broad host ranges. This research further characterizes DLP1 and DLP2 as potential “anti-virulence” phage therapy candidates for the treatment of multidrug resistant bacteria from multiple genera.

The isolation and characterization of Stenotrophomonas maltophilia T4-like bacteriophage DLP6.

Increasing isolation of the extremely antibiotic resistant bacterium Stenotrophomonas maltophilia has caused alarm worldwide due to the limited treatment options available. A potential treatment option for fighting this bacterium is 'phage therapy', the clinical application of bacteriophages to selectively kill bacteria. Bacteriophage DLP6 (vB_SmoM-DLP6) was isolated from a soil sample using clinical isolate S. maltophilia strain D1571 as host. Host range analysis of phage DLP6 against 27 clinical S. maltophilia isolates shows successful infection and lysis in 13 of the 27 isolates tested. Transmission electron microscopy of DLP6 indicates that it is a member of the Myoviridae family. Complete genome sequencing and analysis of DLP6 reveals its richly recombined evolutionary history, featuring a core of both T4-like and cyanophage genes, which suggests that it is a member of the T4-superfamily. Unlike other T4-superfamily phages however, DLP6 features a transposase and ends with 229 bp direct terminal repeats. The isolation of this bacteriophage is an exciting discovery due to the divergent nature of DLP6 in relation to the T4-superfamily of phages.