Sand Fly larvae are capable of positive chemotaxis: a proof of concept study using Phlebotomus papatasi (Diptera: Psychodidae) as a model species.

Phlebotomine sand flies are important vectors of medical and veterinary importance, transmitting pathogens, such as the Leishmania parasites, responsible for 700,000 to 1 million new cases of leishmaniasis every year. The vast majority of the current sand fly surveillance and control tools are tailored against the adult stages, due to the limited knowledge on the ecology of the larval stages. Since vector control is primarily an ecological problem, an in-depth understanding of the behavior of the target insect pests across all the different life stages of their development is required prior to the development of effective control strategies. It is well known that chemical cues play an important role in insect behavior. While there are numerous studies investigating the behavior of adult sand flies in response to chemical sources, there is currently no information available on the response of their larval stages. In this study, novel bioassays were constructed to investigate the effect of chemical cues (gustatory and olfactory) on the behavior of Phlebotomus papatasi (Scopoli) sand fly larvae. The larvae exhibited a clear food preference within a few hours of exposure in a 2-choice bioassay, while, also, demonstrated positive chemotaxis in response to volatile stimuli emitted from their preferred food source. Identification of the specific chemical compounds (or the combination thereof) eliciting attractance response to sand fly immature stages could lead to the development of innovative, and targeted (larval-specific) tools for the surveillance, and management of these important public health pests.

Atypical presentation of mpox in Irrua environs: a case report.

BACKGROUND: Mpox, previously known as monkeypox, -is an orthopoxvirus infection of the skin and previously a public health emergency of international concern. It reemerged in Nigeria over 5 years ago and has since spread to other parts of the world. This is a case report of a confirmed patient who was managed at Irrua Specialist Teaching Hospital, Irrua, Edo State, Nigeria before the global surge. This report shows peculiar differences from previous patients managed at the same center in terms of the relatively prolonged eruptive phase, possible seasonal occurrence of mpox in the community, and some traditional care for mpox and skin rashes. It also corroborates previous reports of possible sexual transmission of mpox in Nigeria before the report from the global outbreak. CASE PRESENTATION: The patient is a 30-year-old Nigerian male artisan with a 2-month history of raised rashes on the body that started on the genitals then involved other parts of the body. There was history of sore throat and unprotected sex with a female partner with similar rash whose other sexual history could not be ascertained. There was also history of "seasonal" rash in his village for about 7 years prior to his symptoms. Examination showed multiple vesicles and some nodules (ulcerating, healing, and healed) on the face, trunk, limbs, gluteal region, scrotum, palms, and sole, an almost circumferential penile ulcer, and lymphadenopathy. Polymerase chain reaction skin samples sent for mpox returned positive, while retroviral and coronavirus disease 2019 screenings were negative. He was managed in isolation while contact tracing in the affected community was initiated. CONCLUSION: Atypical presentations of mpox, as managed in Irrua before the global surge, emphasize the varied spectrum of presentations (typical and atypical) in Nigeria. Therefore, there is a need for a higher index of suspicion for the uncommon presentations which will strengthen case recognition, case management, and community-based interventions as well as surveillance in the prevention and control of mpox in Irrua, its environs, Nigeria, and the world.

Photo-expansion microscopy enables super-resolution imaging of cells embedded in 3D hydrogels.

Hydrogels are extensively used as tunable, biomimetic three-dimensional cell culture matrices, but optically deep, high-resolution images are often difficult to obtain, limiting nanoscale quantification of cell-matrix interactions and outside-in signalling. Here we present photopolymerized hydrogels for expansion microscopy that enable optical clearance and tunable ×4.6-6.7 homogeneous expansion of not only monolayer cell cultures and tissue sections, but cells embedded within hydrogels. The photopolymerized hydrogels for expansion microscopy formulation relies on a rapid photoinitiated thiol/acrylate mixed-mode polymerization that is not inhibited by oxygen and decouples monomer diffusion from polymerization, which is particularly beneficial when expanding cells embedded within hydrogels. Using this technology, we visualize human mesenchymal stem cells and their interactions with nascently deposited proteins at <120 nm resolution when cultured in proteolytically degradable synthetic polyethylene glycol hydrogels. Results support the notion that focal adhesion maturation requires cellular fibronectin deposition; nuclear deformation precedes cellular spreading; and human mesenchymal stem cells display cell-surface metalloproteinases for matrix remodelling.

Experimental Colonization of Sand Flies (Lutzomyia longipalpis; Diptera: Psychodidae) by Bartonella ancashensis.

Background: Bartonella ancashensis is a recently described Bartonella species endemic to Peru, where it causes verruga peruana in humans. While the arthropod vector of B. ancashensis transmission is unknown, human coinfections with Bartonella bacilliformis suggest that phlebotomine sand flies are a vector. Materials and Methods: To address the hypothesis that sand flies are involved in the bacterium's transmission, Lutzomyia longipalpis sand flies were used as an infection model, together with green fluorescent protein-expressing B. ancashensis. Results: Results showed that bacterial infections were clearly established, limited to the anterior midgut of the female fly, and maintained for roughly 7 days. At 3-7 days postinfection, a prominent microcolony of aggregated bacteria was observed in the anterior midgut, immediately distal to the stomodeal valve of the esophagus. In contrast, eggs, diuretic fluid, feces, and other tissues were not infected. Conclusion: These results suggest that certain sand fly species within the endemic zone for B. ancashensis may play a role in the bacterium's maintenance and possibly in its transmission to humans.

Bacteriological Profile and Antibiotic Sensitivity Patterns in Clinical Isolates from the Out-Patient Departments of a Tertiary Hospital in Nigeria.

BACKGROUND: Antimicrobial resistance (AMR) is a rising global public health threat. Knowledge of the circulating pathogens in a particular area and their antibiotic resistance profile is essential to direct clinicians on rational antibiotic prescribing. AIM: The study was conducted to determine the microbial isolates and antibiotic susceptibility profiles of pathogens from a range of clinical samples in a tertiary hospital in Edo Central Senatorial District in Edo State, Nigeria. SETTINGS AND DESIGN: The study was a retrospective analysis of microbiological isolates from clinical specimens collected between January 2016 and December 2019, using standard techniques from outpatient clinic attendees. Chi-square test was used to compare the association of the type of bacterial isolates with patients' sex and level of significance P set as < 0.05. Prevalence rates of bacterial isolates and resistance rates were calculated for each antibiotic used in the microbiological culture. RESULTS: Of the 3,247 clinical specimens processed, 994 (30.6%) showed microbial growth with 436 (43.9%) as gram-positive and 558 (56.1%) as gram-negative bacterial isolates. Escherichia coli (E. coli) made up 286 (28.8%) of all the isolates. Resistance to cotrimoxazole, tetracycline and cloxacilin for gram-poisitive pathogens was 93.1%, 86.4% and 72.5% respectively. For gram-negative pathogens, resistance to amoxycilin, cloxacilin and erythromycin was 100%, 96.9% and 95.6% respectively. Sensitivity to carbapenems, nitrofurantoin, and cefixime was high for gram-negative bacteria (100.0 %,76.8 % and 82.5 % respectively). Gram-positive bacteria exhibited high sensitivity to carbapenems ceftriaxone and cefixime. CONCLUSION: High rates of resistance to common antibiotics were observed for gram-positive and gram-negative isolates. Hospital pharmacies and treatment guidelines should be made to reflect the current patterns of resistance to available antibiotics.

Digital light processing in a hybrid atomic force microscope: In Situ, nanoscale characterization of the printing process.

Stereolithography (SLA) and digital light processing (DLP) are powerful additive manufacturing techniques that address a wide range of applications including regenerative medicine, prototyping, and manufacturing. Unfortunately, these printing processes introduce micrometer-scale anisotropic inhomogeneities due to the resin absorptivity, diffusivity, reaction kinetics, and swelling during the requisite photoexposure. Previously, it has not been possible to characterize high-resolution mechanical heterogeneity as it develops during the printing process. By combining DLP 3D printing with atomic force microscopy in a hybrid instrument, heterogeneity of a single, in situ printed voxel is characterized. Here, we describe the instrument and demonstrate three modalities for characterizing voxels during and after printing. Sensing Modality I maps the mechanical properties of just-printed, resin-immersed voxels, providing the framework to study the relationships between voxel sizes, print exposure parameters, and voxel-voxel interactions. Modality II captures the nanometric, in situ working curve and is the first demonstration of in situ cure depth measurement. Modality III dynamically senses local rheological changes in the resin by monitoring the viscoelastic damping coefficient of the resin during patterning. Overall, this instrument equips researchers with a tool to develop rich insight into resin development, process optimization, and fundamental printing limits.

Injury-mediated stiffening persistently activates muscle stem cells through YAP and TAZ mechanotransduction.

The skeletal muscle microenvironment transiently remodels and stiffens after exercise and injury, as muscle ages, and in myopathic muscle; however, how these changes in stiffness affect resident muscle stem cells (MuSCs) remains understudied. Following muscle injury, muscle stiffness remained elevated after morphological regeneration was complete, accompanied by activated and proliferative MuSCs. To isolate the role of stiffness on MuSC behavior and determine the underlying mechanotransduction pathways, we cultured MuSCs on strain-promoted azide-alkyne cycloaddition hydrogels capable of in situ stiffening by secondary photocrosslinking of excess cyclooctynes. Using pre- to post-injury stiffness hydrogels, we found that elevated stiffness enhances migration and MuSC proliferation by localizing yes-associated protein 1 (YAP) and WW domain-containing transcription regulator 1 (WWTR1; TAZ) to the nucleus. Ablating YAP and TAZ in vivo promotes MuSC quiescence in postinjury muscle and prevents myofiber hypertrophy, demonstrating that persistent exposure to elevated stiffness activates mechanotransduction signaling maintaining activated and proliferating MuSCs.

No Evidence of rVSV-Ebola Virus Vaccine Replication or Dissemination in the Sand Fly Phlebotomus papatasi.

Following vaccination with the live attenuated, recombinant vesicular stomatitis virus Indiana serotype Ebola virus (rVSV-EBOV) vaccine, persons may exhibit a transient vaccine-associated viremia. To investigate the potential for Old World sand flies to transmit this vaccine following feeding on a viremic person, we fed laboratory-reared Phlebotomus papatasi an artificial blood meal containing 7.2 log10 plaque-forming units of rVSV-EBOV. Replication or dissemination was not detected in the body or legs of any P. papatasi collected at seven (n = 75) or 15 (n = 75) days post-feed. These results indicate a low potential for rVSV-EBOV to replicate and disseminate in P. papatasi, a species whose geographic distribution ranges from Morocco to southwest Asia and as far north as southern Europe.

Antibiotic Self-Medication among Adult Out-patient Clinic Attendees in Nigeria.

BACKGROUND AND OBJECTIVES: The study was conducted to determine the prevalence and pattern of antibiotic selfmedication and associated risk factors among out-patients clinic attendees in Edo state, Nigeria. METHODS: A cross-sectional study was conducted among 800 consenting adult attendees at the general out -patient department of five secondary and one tertiary health facilities. Data were analyzed using Statistical Package for Social Sciences. Chi-square test was used for bivariate analysis, and significant variables analyzed with multivariate logistic regression. Statistical significance p was set at < 0.05. RESULTS: Three hundred and seventy four (46.8%) of 800 respondents had self-medicated with antibiotics 6 months preceding the study with commonly used drugs as Ampicillin/Cloxacillin (31.3%) and Amoxycillin (24.8%). Respiratory tract conditions were the most common reasons for taking antibiotics, 100 (27.2%). Primary reasons for self-medication was the availability of the drug at the local drug store. Being divorced or widowered (AOR 0.302, 95% CI 0.117-0.781, p = 0.01) and age > 70 years (AOR 0.18, 95% CI 0.04-0.84, p = 0.03) were significantly negatively associated with antibiotic self-medication. CONCLUSION: The practice of self-medication with antibiotics was high among adult out-patients in the study area, and primarily due to the ease with which antibiotics can be brought across the counter. Restrictions on over-the-counter sale of antibiotics and sensitization of drug sellers is an urgently required intervention to stem the tide. Public enlightenment on dangers of self-medication especially targeting at risk groups identified by the study is also required.

Defining the Cardiac Fibroblast Secretome in a Fibrotic Microenvironment.

Background Cardiac fibroblasts (CFs) have the ability to sense stiffness changes and respond to biochemical cues to modulate their states as either quiescent or activated myofibroblasts. Given the potential for secretion of bioactive molecules to modulate the cardiac microenvironment, we sought to determine how the CF secretome changes with matrix stiffness and biochemical cues and how this affects cardiac myocytes via paracrine signaling. Methods and Results Myofibroblast activation was modulated in vitro by combining stiffness cues with TGFß1 (transforming growth factor ß 1) treatment using engineered poly (ethylene glycol) hydrogels, and in vivo with isoproterenol treatment. Stiffness, TGFß1, and isoproterenol treatment increased AKT (protein kinase B) phosphorylation, indicating that this pathway may be central to myofibroblast activation regardless of the treatment. Although activation of AKT was shared, different activating cues had distinct effects on downstream cytokine secretion, indicating that not all activated myofibroblasts share the same secretome. To test the effect of cytokines present in the CF secretome on paracrine signaling, neonatal rat ventricular cardiomyocytes were treated with CF conditioned media. Conditioned media from myofibroblasts cultured on stiff substrates and activated by TGFß1 caused hypertrophy, and one of the cytokines in that media was insulin growth factor 1, which is a known mediator of cardiac myocyte hypertrophy. Conclusions Culturing CFs on stiff substrates, treating with TGFß1, and in vivo treatment with isoproterenol all caused myofibroblast activation. Each cue had distinct effects on the secretome or genes encoding the secretome, but only the secretome of activated myofibroblasts on stiff substrates treated with TGFß1 caused myocyte hypertrophy, most likely through insulin growth factor 1.

The Effect of Thiol Structure on Allyl Sulfide Photodegradable Hydrogels and their Application as a Degradable Scaffold for Organoid Passaging.

Intestinal organoids are useful in vitro models for basic and translational studies aimed at understanding and treating disease. However, their routine culture relies on animal-derived matrices that limit translation to clinical applications. In fact, there are few fully defined, synthetic hydrogel systems that allow for the expansion of intestinal organoids. Here, an allyl sulfide photodegradable hydrogel is presented, achieving rapid degradation through radical addition-fragmentation chain transfer (AFCT) reactions, to support routine passaging of intestinal organoids. Shear rheology to first characterize the effect of thiol and allyl sulfide crosslink structures on degradation kinetics is used. Irradiation with 365 nm light (5 mW cm-2 ) in the presence of a soluble thiol (glutathione at 15 × 10-3 m), and a photoinitiator (lithium phenyl-2,4,6-trimethylbenzoylphosphinate at 1 × 10-3 m), leads to complete hydrogel degradation in less than 15 s. Allyl sulfide hydrogels are used to support the formation of epithelial colonies from single intestinal stem cells, and rapid photodegradation is used to achieve repetitive passaging of stem cell colonies without loss in morphology or organoid formation potential. This platform could support long-term culture of intestinal organoids, potentially replacing the need for animal-derived matrices, while also allowing systematic variations to the hydrogel properties tailored for the organoid of interest.

Relaxation of Extracellular Matrix Forces Directs Crypt Formation and Architecture in Intestinal Organoids.

Intestinal organoid protocols rely on the use of extracellular scaffolds, typically Matrigel, and upon switching from growth to differentiation promoting media, a symmetry breaking event takes place. During this stage, the first bud like structures analogous to crypts protrude from the central body and differentiation ensues. While organoids provide unparalleled architectural and functional complexity, this sophistication is also responsible for the high variability and lack of reproducibility of uniform crypt-villus structures. If function follows form in organoids, such structural variability carries potential limitations for translational applications (e.g., drug screening). Consequently, there is interest in developing synthetic biomaterials to direct organoid growth and differentiation. It has been hypothesized that synthetic scaffold softening is necessary for crypt development, and these mechanical requirements raise the question, what compressive forces and subsequent relaxation are necessary for organoid maturation? To that end, allyl sulfide hydrogels are employed as a synthetic extracellular matrix mimic, but with photocleavable bonds that temporally regulate the material's bulk modulus. By varying the extent of matrix softening, it is demonstrated that crypt formation, size, and number per colony are functions of matrix softening. An understanding of the mechanical dependence of crypt architecture is necessary to instruct homogenous, reproducible organoids for clinical applications.

Extended Exposure to Stiff Microenvironments Leads to Persistent Chromatin Remodeling in Human Mesenchymal Stem Cells.

Bone marrow derived human mesenchymal stem cells (hMSCs) are a promising cell source for regenerative therapies; however, ex vivo expansion is often required to achieve clinically useful cells numbers. Recent results reveal that when MSCs are cultured in stiff microenvironments, their regenerative capacity can be altered in a manner that is dependent on time (e.g., a mechanical dosing analogous to a chemical one). It is hypothesized that epigenomic modifications are involved in storing these mechanical cues, regulating gene expression, and ultimately leading to a mechanical memory. Using hydrogels containing an allyl sulfide cross-linker and a radical-mediated addition-fragmentation chain transfer process, in situ softened hMSC-laden hydrogels at different time points are achieved and the effects of short-term and long-term mechanical dosing on epigenetic modifications in hMSCs are quantified. Results show that histone acetylation and chromatin organization adapt rapidly after softening and can be reversible or irreversible depending on time of exposure to stiff microenvironments. Furthermore, epigenetic modulators are differentially expressed depending on the culture history. Collectively, these experiments suggest that epigenetic remodeling can be persistent and might be a memory keeper.

Metagenomic sequencing at the epicenter of the Nigeria 2018 Lassa fever outbreak.

The 2018 Nigerian Lassa fever season saw the largest ever recorded upsurge of cases, raising concerns over the emergence of a strain with increased transmission rate. To understand the molecular epidemiology of this upsurge, we performed, for the first time at the epicenter of an unfolding outbreak, metagenomic nanopore sequencing directly from patient samples, an approach dictated by the highly variable genome of the target pathogen. Genomic data and phylogenetic reconstructions were communicated immediately to Nigerian authorities and the World Health Organization to inform the public health response. Real-time analysis of 36 genomes and subsequent confirmation using all 120 samples sequenced in the country of origin revealed extensive diversity and phylogenetic intermingling with strains from previous years, suggesting independent zoonotic transmission events and thus allaying concerns of an emergent strain or extensive human-to-human transmission.

Secondary Photocrosslinking of Click Hydrogels To Probe Myoblast Mechanotransduction in Three Dimensions.

Muscle cells sense the mechanical properties of their microenvironment, and these properties can change in response to injury or disease. Hydrogels with dynamic material properties can be used to study the effect of such varying mechanical signals. Here, we report the ability of azadibenzocyclooctyne to undergo a cytocompatible, photoinitiated crosslinking reaction. This reaction is exploited as a strategy for on-demand stiffening of three-dimensional cell scaffolds formed through an initial strain-promoted azide-alkyne cycloaddition. Myoblasts encapsulated in these networks respond to increased matrix stiffness through decreased cell spreading and nuclear localization of Yes-associated protein 1 (YAP). However, when the photocrosslinking reaction is delayed to allow cell spreading, elongated myoblasts display increased YAP nuclear localization.

A Reversible and Repeatable Thiol-Ene Bioconjugation for Dynamic Patterning of Signaling Proteins in Hydrogels.

Biomolecule-functionalized hydrogels have emerged as valuable cell culture platforms to recapitulate the mechanical and biochemical properties of the extracellular niche. The typical strategy to functionalize hydrogels with biomolecules involves directly tethering them to the hydrogel backbone resulting in a static material. Thus, this approach fails to capture the dynamic changes in biomolecule composition that occur during biological processes or that may be required for regenerative medicine applications. Moreover, it also limits the scope of biomolecules to simple peptides, as signaling proteins generally have poor stability under cell culture conditions and lose their bioactivity over time. To that end, we sought to develop a bioconjugation reaction that would enable reversible and repeatable tethering of signaling proteins to hydrogels, so that spent protein could be released on-demand and replaced with fresh protein as needed. Specifically, we designed an allyl sulfide chain-transfer agent that enables a reversible, photomediated, thiol-ene bioconjugation of signaling proteins to hydrogels. Upon addition of a thiolated protein to the allyl sulfide moiety, the previously tethered protein is released, and the "ene" functionality is regenerated. Using this approach, we demonstrate that protein patterning can be achieved in hydrogels through a thiol-ene reaction, and the patterned protein can then be released through a subsequent thiol-ene reaction of a PEG thiol. Importantly, this process is repeatable through multiple iterations and proceeds at physiologically relevant signaling protein concentrations. Finally, we demonstrate that whole signaling proteins can be patterned and released in the presence of cells, and that cells respond to their presentation with spatial fidelity. Combined, these data represent the first example of a methodology that enables fully reversible and repeatable patterning and release of signaling proteins from hydrogels.

Photopolymerized dynamic hydrogels with tunable viscoelastic properties through thioester exchange.

The extracellular matrix (ECM) constitutes a viscoelastic environment for cells. A growing body of evidence suggests that the behavior of cells cultured in naturally-derived or synthetic ECM mimics is influenced by the viscoelastic properties of these substrates. Adaptable crosslinking strategies provide a means to capture the viscoelasticity found in native soft tissues. In this work, we present a covalent adaptable hydrogel based on thioester exchange as a biomaterial for the in vitro culture of human mesenchymal stem cells. Through control of pH, gel stoichiometry, and crosslinker structure, viscoelastic properties in these crosslinked networks can be modulated across several orders of magnitude. We also propose a strategy to alter these properties in existing networks by the photo-uncaging of the catalyst 4-mercaptophenylacetic acid. Mesenchymal stem cells encapsulated in thioester hydrogels are able to elongate in 3D and display increased proliferation relative to those in static networks.

Synthesis of microgel sensors for spatial and temporal monitoring of protease activity.

Proteases are involved in almost every important cellular activity, from embryonic morphogenesis to apoptosis. To study protease activity in situ, hydrogels provide a synthetic mimic of the extracellular matrix (ECM) and have utility as a platform to study activity, such as those related to cell migration, in three-dimensions. While 3-dimensional visualization of protease activity could prove quite useful to elucidate the proteolytic interaction at the interface between cells and their surrounding environment, there has been no versatile tool to visualize local proteolytic activity in real time. Here, micron-sized gels were synthesized by inverse suspension polymerization using thiolene photo-click chemistry. The size distribution was selected to avoid cellular uptake and to lower cytotoxicity, while simultaneously allowing the integration of peptide-based FRET sensors of local cell activity. Proteolytic activity of collagenase was detected within an hour via changes in fluorescence of embedded microgels; incubation of microgel sensors with A375 melanoma cells showed upregulated MMP activity in the presence of soluble fibronectins in media. The microgel sensors were readily incorporated into both gelatin and poly(ethylene glycol) (PEG) hydrogels and used to successfully detect spatiotemporal proteolytic activity of A375 melanoma cells. Finally, a tumor model was constructed from a hydrogel microwell array that was used to aggregate A375 melanoma cells, and local variations in proteolytic activity were monitored as a function of distance from the cell aggregate center.