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.

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.

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.

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.

Spatiotemporal hydrogel biomaterials for regenerative medicine.

Hydrogels mimic many of the physical properties of soft tissue and are widely used biomaterials for tissue engineering and regenerative medicine. Synthetic hydrogels have been developed to recapitulate many of the healthy and diseased states of native tissues and can be used as a cell scaffold to study the effect of matricellular interactions in vitro. However, these matrices often fail to capture the dynamic and heterogenous nature of the in vivo environment, which varies spatially and during events such as development and disease. To address this deficiency, a variety of manufacturing and processing techniques are being adapted to the biomaterials setting. Among these, photochemistry is particularly well suited because these reactions can be performed in precise three-dimensional space and at specific moments in time. This spatiotemporal control over chemical reactions can also be performed over a range of cell- and tissue-relevant length scales with reactions that proceed efficiently and harmlessly at ambient conditions. This review will focus on the use of photochemical reactions to create dynamic hydrogel environments, and how these dynamic environments are being used to investigate and direct cell behavior.

Assessment of knowledge and sexual behaviour among undergraduates in a Nigerian tertiary institution.

Background and Introduction: The achievement of the Millennium Development Goals (MDGs), particularly goals 5 and 6, is strongly underpinned by the progress that can be made on sexual and reproductive health education of young adults. The large population of young single adults in tertiary institutions in Nigeria and the present dearth of human resources for health necessitates that interventions for this group of individuals are tailored to meet identified gaps in awareness and sexual behaviour. This study sets out to assess the sexual behaviour and practice among single-undergraduates in a tertiary institution in Nigeria. Method: A descriptive cross sectional study was carried out among 410 consenting students selected through a multistage sampling method. Data was collected using a structured self- administered questionnaire and analysed using Statistical Package for Scientific Solutions (SPSS) version 16.0. Results: Mean age of respondents was 20 + 4.4 years, 228 (55.6%) were females and 182 (44.4%) males. The respondents' awareness of contraceptive devices was 81.7% (335) for condoms and less than 20% knew about other forms of contraception such as intrauterine device, tubal ligation and vasectomy. Over 65.7% (270) opined abstinence and use of condom to be the ideal methods for prevention of HIV/AIDS/ STI transmission. The overall mean age at first sexual contact was 14.0+1.4((14.2+1.6) in males and (13.8 +1.2) in females). A Very high proportion of sexually active respondents (93.6%) volunteered they do not routinely use condom in their sexual encounter. Undesired pregnancies occurred in about 11% of females. Recommendations: There is an urgent need for the establishment of specially designated youth friendly centres in the tertiary institutions in the country, manned with staff appropriately trained in the delivery of reproductive health information and services.

Engaging Market Traders in Lassa Fever Campaign: Assessment of Knowledge and Risk Behaviour.

BACKGROUND: Markets provide a forum for reaching a large adult population with information on Lassa fever, and therefore understanding the food handling practices of traders may provide the foundation for an effective campaign against Lassa fever. This study was undertaken to provide baseline information on knowledge and food handling practices of traders in local markets in a Lassa fever endemic state of Nigeria. STUDY DESIGN: A structured questionnaire was used to obtain food handling habits that facilitate the transmission of Lassa virus from a cross sectional study involving 385 traders in three major markets in Edo state and data analyzed using SPSS version 15. RESULTS: Two hundred and ninety three (76.1%) had ever heard of Lassa fever, 27 (9.2%) had good knowledge. Good knowledge was significantly associated with higher educational status (p < 0.00) and male gender (p=0.03). Thirty seven (12.6%) respondents sun-dried their food frequently, 105 (35.8%) stored utensils in rodent proof containers, and 136 (46.4%) had the habit of eating garri soaked in water. One hundred and ninety (49.4%) respondents had food hygiene practices that were favorable for spread of Lassa fever. CONCLUSION: The observed gaps in knowledge of Lassa fever and food hygiene may be addressed through tailored health messages. In this way, market campaigns will be effective in increasing knowledge of Lassa fever, and traders can themselves become peer educators.

Knowledge of Secondary School Children in Edo State on Lassa Fever and its Implications for Prevention and Control.

BACKGROUND: Seasonal outbreaks of Lassa fever in West Africa cause significant morbidity and mortality across all ages. In addition to present efforts to raise awareness, school children can be targeted to become peer and family health educators. The study was carried out to determine the knowledge of Lassa fever among secondary school children, and household practices that increase risk of the infection. STUDY DESIGN: In a cross sectional survey, 561 secondary school students randomly selected from schools in Edo State were interviewed by means of a self - administered questionnaire that sought information on knowledge of Lassa fever and practices within the home that favour rodent contact . Data were analyzed using Statistical Package for Social Sciences version 15. RESULTS: Knowledge of Lassa fever was poor among 259 (49.4%) respondents, fair in 216 (41.2%) and good in 49 (9.4%). Female gender (< 0.01), monogamous family structure (p < 0.04) , and being in senior secondary class ( p=0.01) were predictors of high knowledge score. Self- reported Lassa fever risk conditions were found to be of low prevalence in 311(55.4%) and high in 250 (44.6%) homes, and associated with educational status of mother ( p=0.00) and father, (p =0.00). CONCLUSION: School children in endemic communities lack good knowledge of Lassa fever, but when properly guided, have the potential to become peer and family educators.

Design and characterization of a synthetically accessible, photodegradable hydrogel for user-directed formation of neural networks.

Hydrogels with photocleavable units incorporated into the cross-links have provided researchers with the ability to control mechanical properties temporally and study the role of matrix signaling on stem cell function and fate. With a growing interest in dynamically tunable cell culture systems, methods to synthesize photolabile hydrogels from simple precursors would facilitate broader accessibility. Here, a step-growth photodegradable poly(ethylene glycol) (PEG) hydrogel system cross-linked through a strain promoted alkyne-azide cycloaddition (SPAAC) reaction and degraded through the cleavage of a nitrobenzyl ether moiety integrated into the cross-links is developed from commercially available precursors in three straightforward synthetic steps with high yields (>95%). The network evolution and degradation properties are characterized in response to one- and two-photon irradiation. The PEG hydrogel is employed to encapsulate embryonic stem cell-derived motor neurons (ESMNs), and in situ degradation is exploited to gain three-dimensional control over the extension of motor axons using two-photon infrared light. Finally, ESMNs and their in vivo synaptic partners, myotubes, are coencapsulated, and the formation of user-directed neural networks is demonstrated.

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.

Prevalence of urinary schistosomiasis among school children in a rural community in South-South, Nigeria.

BACKGROUND: Schistosomiasis is a major health problems in tropical Africa with school age children being most affected. The study aimed to assess the prevalence of the disease among primary and secondary school children in a rural community in Edo State. STUDY DESIGN: A cross sectional study was carried out in a rural community in Etsako West local government area of Edo state. Respondents included all primary and secondary school students in the community. A structured administered questionnaire and urine microscopy were used for data collection. RESULTS AND CONCLUSION: Twenty-four percent of subjects reported passing blood in urine out of which 10.9% had ever sought medical attention for the haematuria. The overall prevalence of urinary Schistosomiasis, as confirmed by the presence of eggs of Schistosoma haematobium was 10.7% with males being almost 3 times as many as the affected females; 32 (74.4%) versus 11(25.6%). Prevalence of microhaematuria was 4.96 %, and protenuria 7.94% . There is need for an urgent mapping of communities served by the infested stream, as well as enlightenment campaigns to educate the rural populace on the disease and it's complications.

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.

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.

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.

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.

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.

A case of Campylobacter jejuni bacteremia in a renal transplant patient.

Only 2 cases of Campylobacter bacteremia have been reported in renal transplant recipients, to our knowledge, with both resulting in significant morbidity and mortality. We present a case of a 56-year-old renal transplant recipient who presented with brief diarrheal illness followed by Campylobacter jejuni bacteremia. She remained asymptomatic for 5 days after initial presentation despite positive blood cultures. She was treated with levofloxacin for a total of 4 weeks and, fortunately, did not develop any complications. C. jejuni should be considered in the differential diagnosis as a potential cause of bacteremia in immunosuppressed renal transplant patients presenting with diarrheal illness.

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.

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.