Carnivore Protoparvovirus-1 Associated With an Outbreak of Hemorrhagic Gastroenteritis in Small Indian Civets.

Carnivore protoparvovirus-1 (CPPV-1) infection has been reported frequently in both domestic and wildlife species including wild carnivores. Fifty-five captive small Indian civets (Viverricula indica), farmed for perfume production in Eastern Thailand, showed clinical signs of acute bloody diarrhea, anorexia, vomiting, circling, and seizures. The disease spread within the farm and resulted in the death of 38 of the 55 civets (69% mortality) within a month. Fecal swabs were collected from the 17 surviving civets, and necropsy was performed on 7 of the dead civets. Pathologic findings were severe hemorrhagic gastroenteritis with generalized lymphadenopathy. CPPV-1 was identified in both fecal swabs and postmortem samples by species-specific polymerase chain reaction. Further whole-gene sequencing and restriction fragment length polymorphism analysis suggested feline panleukopenia virus (FPV) as the causative agent. The viral tropism and tissue distribution were confirmed by immunohistochemistry, with immunolabeling in the cytoplasm and nucleus of small intestinal crypt epithelial cells, villous enterocytes, histiocytes in lymphoid tissues, myenteric nerve plexuses, and cerebral and cerebellar neurons. Phylogenetic analysis of civet-derived CPPV-1 indicated a genetic similarity close to the FPV HH-1/86 strain detected in a jaguar (Panthera onca) in China. To our knowledge, this mass die-off of civets is the first evidence of disease associated with CPPV-1 infection in the subfamily Viverrinae. These findings support the multi-host range of parvovirus infection and raises awareness for CPPV-1 disease outbreaks in wildlife species.

Feline Morbillivirus Infection Associated With Tubulointerstitial Nephritis in Black Leopards (Panthera pardus).

Feline morbillivirus (FeMV) is an emerging RNA virus in the Paramyxoviridae family that was recently discovered in domestic cats (Felis catus). To date, 2 genotypes (FeMV-1 and FeMV-2) have been detected in cats from various countries, and FeMV-1 is recognized as a pathogen associated with nephritis. However, information regarding the pathological roles and potential transmission to other felids is limited. In this article, we describe the identification of FeMV in 2 black leopards (Panthera pardus) in Thailand that showed severe azotemia and tubulointerstitial nephritis. Molecular analysis of the partial coding sequence of the L gene revealed that these leopard FeMV strains were genetically close to the FeMV-1 isolate from domestic Thai cats. Immunohistochemistry and immunofluorescence analyses using polyclonal IgG antibodies against the FeMV matrix (M) protein showed FeMV-M antigen in renal tubular epithelial cells. These analyses also showed infiltrating lymphocytes in the renal parenchymal lesions and in the cytoplasm of lymphoid cells residing in the spleen, suggesting viral tropism and a possible pathological role. These findings are the first evidence that indicates that the black leopard could be a possible host for FeMV infection. As for other cats, the role of FeMV as a potential cause of renal disease remains to be established. The pathogenesis of FeMV infection in black leopards, or in other wild felids, through a viral transmission mechanism warrants further investigation.

Epizootic of multi-centric, squamous cell carcinomas in populations of Indo-Pacific humpbacked dolphins Sousa chinensis in Thai waters.

Over the span of several years, 3 Indo-Pacific humpbacked dolphins died and were necropsied in Thailand. These 3 animals were all captive-bred at Oasis Sea World (Chanthaburi, Thailand), and displayed similar macroscopic progressive cutaneous lesions diagnosed as squamous cell carcinomas. In 2 of the 3 animals, necropsy revealed a severe fibrinosuppurative tracheitis and pneumonia secondary to metastasis of a cutaneous squamous cell carcinoma which extended from the head throughout the trunk and flippers. The tumors were characterized by coalescing botryoid masses with severe areas of cutaneous erosion, ulceration and necrohemorrhagic dermatitis. There was evidence of metastasis to the lungs and hilar lymph nodes. Necropsy of the third animal revealed similar progressive cutaneous squamous cell carcinomas but without evidence of metastasis. DNA molecular analysis of homogenized neoplastic tissue was conducted using polymerase chain reaction for both herpesvirus and papillomavirus in 2 of the 3 cases. In the first case, the tissues were positive for a herpesvirus alone, and this was phylogenetically classified as an alphaherpesvirus. This new herpesvirus has been tentatively named Sousa chinensis alphaherpesvirus. The second animal was negative for this novel herpesvirus and the third was not analyzed. In addition to the captive population, there is photographic evidence from 2 separate wild populations of Indo-Pacific humpbacked dolphins in the Gulf of Thailand, of a macroscopically identical proliferative and ulcerative process suspected to be squamous cell carcinomas.

Canine Bocavirus Type 2 Infection Associated With Intestinal Lesions.

Bocaviruses are small nonenveloped DNA viruses belonging to the Bocaparvovirus genus of the Parvoviridae family and have been linked to both respiratory and enteric disease in humans and animals. To date, 3 bocaviruses, canine bocaviruses 1 to 3 (CBoV-1-3), have been shown to affect dogs with different disease manifestations reported for infected animals. We used next-generation sequencing to identify a novel strain of canine CBoV-2 (CBoV TH-2016) in a litter of puppies that died in Thailand from acute dyspnea and hemoptysis, for which no causal pathogen could be identified in routine assays. Analysis of the complete coding sequences of CBoV TH-2016 showed that this virus was most closely related to a strain previously identified in South Korea (isolate 14D193), with evidence of genetic recombination in the VP2 gene with related strains from South Korea and Hong Kong. Use of quantitative polymerase chain reaction showed the presence of CBoV TH-2016 in several tissues, suggesting hematogenous virus spread, while only intestinal tissue was found to be positive by in situ hybridization and electron microscopy. Histologic small intestinal lesions associated with CBoV TH-2016 infection were eosinophilic intranuclear inclusion bodies within villous enterocytes without villous atrophy or fusion, similar to those previously considered pathognomonic for CBoV-1 infection. Therefore, this study provides novel insights in the pathogenicity of canine bocavirus infections and suggests that a novel recombinant CBoV-2 may result in atypical findings of CBoV infection. Although the specific cause of death of these puppies remained undetermined, a contributory role of enteric CBoV TH-2016 infection is possible.

Bringing macromolecules into cells and evading endosomes by oxidized carbon nanoparticles.

A great challenge exists in finding safe, simple, and effective delivery strategies to bring matters across cell membrane. Popular methods such as viral vectors, positively charged particles and cell penetrating peptides possess some of the following drawbacks: safety issues, lysosome trapping, limited loading capacity, and toxicity, whereas electroporation produces severe damages on both cargoes and cells. Here, we show that a serendipitously discovered, relatively nontoxic, water dispersible, stable, negatively charged, oxidized carbon nanoparticle, prepared from graphite, could deliver macromolecules into cells, without getting trapped in a lysosome. The ability of the particles to induce transient pores on lipid bilayer membranes of cell-sized liposomes was demonstrated. Delivering 12-base-long pyrrolidinyl peptide nucleic acids with d-prolyl-(1S,2S)-2-aminocyclopentanecarboxylic acid backbone (acpcPNA) complementary to the antisense strand of the NF-κB binding site in the promoter region of the Il6 gene into the macrophage cell line, RAW 264.7, by our particles resulted in an obvious accumulation of the acpcPNAs in the nucleus and decreased Il6 mRNA and IL-6 protein levels upon stimulation. We anticipate this work to be a starting point in a new drug delivery strategy, which involves the nanoparticle that can induce a transient pore on the lipid bilayer membrane.

Clinical, radiographic, and histologic analysis of the effects of acemannan used in direct pulp capping of human primary teeth: short-term outcomes.

Acemannan has been previously reported as a direct pulp-capping agent in animal study. This natural material demonstrated its biocompatibility and enhanced reparative dentin formation. The objective of this study was to investigate the action of acemannan as a direct pulp-capping material in human primary teeth with deep caries. Forty-two deeply carious mandibular primary molars from 37 children, aged 7-11 years old diagnosed with reversible pulpitis were studied. After completely removing the infected dentine, teeth with a pinpoint pulpal exposure were randomly divided into two treatment groups: acemannan or calcium hydroxide. A glass-ionomer cement base was applied to all teeth prior to restoration with stainless steel crowns. Clinical and radiographic evaluation was performed 6 months post-treatment. The teeth due to exfoliate were extracted and histopathologically evaluated for inflammation, dentine bridge formation, and soft tissue organization. At 6 months, the overall clinical and radiographic success rates of direct pulp capping with acemannan and calcium hydroxide at 6 months were 72.73 and 70.0 %, respectively. The histopathological results indicated that the acemannan-treated group had significantly better histopathological responses compared with the calcium hydroxide-treated group (p < 0.05). These data suggest acemannan offers a valuable alternative biomaterial for vital pulp therapy in primary teeth.

Acrylate-tethering drug carrier: covalently linking carrier to biological surface and application in the treatment of Helicobacter pylori infection.

The development of carriers to sustain drugs at stomach surface is an attractive strategy to increase drug bioavailability locally and systematically. So far, the only reported carrier that can form a covalent bond with mucus, the thiolated carrier, relies on a reversible disulfide exchange reaction between thiols on the carrier and disulfide bridges on the mucus. Here we show the design and fabrication of a cellulose carrier with tethering acrylate groups (denoted here as clickable carrier) that, under a nontoxic condition, can efficiently react with thiols on biomaterials in situ through the thermodynamically driven and kinetically probable Michael thiol-ene click reaction. Here we show the attachments of the clickable carriers to a mucin protein, a surface of human laryngeal carcinoma cells, and a surface of a fresh porcine stomach. We also show that the required thiol moieties can be generated in situ by reducing existing cystine disulfide bridges with either the edible vitamin C or the relatively nontoxic tris(2-carboxyethyl) phosphine. Comparing to a control carrier, the clickable carrier can increase some drug concentrations in an ex vivo stomach tissue, and improve the Helicobacter pylori treatment in infected C57BL/6 mice.

Effect of acemannan, an extracted polysaccharide from Aloe vera, on BMSCs proliferation, differentiation, extracellular matrix synthesis, mineralization, and bone formation in a tooth extraction model.

Aloe vera is a traditional wound healing medicine. We hypothesized acemannan, a polysaccharide extracted from Aloe vera gel, could affect bone formation. Primary rat bone marrow stromal cells (BMSCs) were treated with various concentrations of acemannan. New DNA synthesis, VEGF, BMP-2, alkaline phosphatase activity, bone sialoprotein, osteopontin expression, and mineralization were determined by [(3)H] thymidine incorporation assay, ELISA, biochemical assay, western blotting, and Alizarin Red staining, respectively. In an animal study, mandibular right incisors of male Sprague-Dawley rats were extracted and an acemannan treated sponge was placed in the socket. After 1, 2, and 4 weeks, the mandibles were dissected. Bone formation was evaluated by dual-energy X-ray absorptiometry and histopathological examination. The in vitro results revealed acemannan significantly increased BMSC proliferation, VEGF, BMP-2, alkaline phosphatase activity, bone sialoprotein and osteopontin expression, and mineralization. In-vivo results showed acemannan-treated groups had higher bone mineral density and faster bone healing compared with untreated controls. A substantial ingrowth of bone trabeculae was observed in acemannan-treated groups. These data suggest acemannan could function as a bioactive molecule inducing bone formation by stimulating BMSCs proliferation, differentiation into osteoblasts, and extracellular matrix synthesis. Acemannan could be a candidate natural biomaterial for bone regeneration.

Kaempferia parviflora Extracellular Vesicle Loaded with Clarithromycin for the Treatment of Helicobacter pylori Infection.

Purpose: Kaempferia parviflora extracellular vesicles (KPEVs) have been reported as promising nanovesicles for drug delivery. This study aimed to load clarithromycin (CLA) into KPEVs (KPEVS-CLA) and determine the physical properties, drug-releasing efficiency, gastric cell uptake, anti-H. pylori activities, and anti-inflammatory responses in comparison with free CLA and KPEVs. Methods: The size and surface charge of KPEVs-CLA were evaluated using dynamic light scattering and visualized using a transmission electron microscope. The encapsulation efficiency (EE%), loading capacity (LC%), and drug release of KPEVs-CLA were examined using HPLC. Anti-H. pylori growth and anti-adhesion were evaluated. IL-8 gene expression, NF-κB signaling proteins, and anti-inflammatory profiles were examined using qRT-PCR, Western blotting, and Bio-Plex immunoassay, respectively. Anti-chemotaxis was then examined using a Transwell assay. Results: KPEVs-CLA were intact and showed a negative surface charge similar to that of KPEVs. However, slightly enlarged KPEVs were observed. CLA was successfully loaded into KPEVs with EE of 93.45% ± 2.43%, LC of 9.3% ± 3.02%. CLA release in the PBS and gastric mimic buffer with Fickian diffusion (n ≤ 0.43) according to Korsmeyer-Peppas kinetic model (R2=0.98). KPEVs-CLA was localized in the gastric cells' cytoplasm and perinuclear region. Anti-H. pylori growth and anti-H. pylori adhesion of KPEVs-CLA were compared with those of free CLA with no cytotoxicity to adenocarcinoma gastric cells. KPEVs-CLA significantly reduced IL-8, G-CSF, MIP-1α, and MIP-1ß levels. Moreover, KPEVs-CLA showed a superior effect over CLA in reducing G-CSF, MIP-1α, and NF-κB phosphorylation and monocyte chemotactic activities. Conclusion: KPEVs serve as potential carriers of CLA. They exhibited a higher efficiency in inhibiting gastric cell inflammation mediated by H. pylori infection than free CLA. The establishment of KPEVs-CLA as a nanodrug delivery model for H. pylori treatment could be applied to other plant extracellular vesicles or loaded with other cancer drugs for gastric cancer treatment.

Dissolvable microneedles in the skin: Determination the impact of barrier disruption and dry skin on dissolution.

Dissolvable microneedles (DMNs), fabricated from biocompatible materials that dissolve in both water and skin have gained popularity in dermatology. However, limited research exists on their application in compromised skin conditions. This study compares the hyaluronic acid-based DMNs penetration, formation of microchannels, dissolution, and diffusion kinetics in intact, barrier-disrupted (tape stripped), and dry (acetone-treated) porcine ear skin ex vivo. After DMNs application, comprehensive investigations including dermoscopy, stereomicroscope, skin hydration, transepidermal water loss (TEWL), optical coherence tomography (OCT), reflectance confocal laser scanning microscopy (RCLSM), confocal Raman micro-spectroscopy (CRM), two-photon tomography combined with fluorescence lifetime imaging (TPT-FLIM), histology, and scanning electron microscopy (SEM) were conducted. The 400 µm long DMNs successfully penetrated the skin to depths of ≈200 µm for dry skin and ≈200-290 µm for barrier-disrupted skin. Although DMNs fully inserted into all skin conditions, their dissolution rates were high in barrier-disrupted and low in dry skin, as observed through stereomicroscopy and TPT-FLIM. The dissolved polymer exhibited a more significant expansion in barrier-disrupted skin compared to intact skin, with the smallest increase observed in dry skin. Elevated TEWL and reduced skin hydration levels were evident in barrier-disrupted and dry skins compared to intact skin. OCT and RCLSM revealed noticeable skin indentation and pronounced microchannel areas, particularly in barrier-disrupted and dry skin. Additional confirmation of DMN effects on the skin and substance dissolution was obtained through histology, SEM, and CRM techniques. This study highlights the impact of skin condition on DMN effectiveness, emphasizing the importance of considering dissolvability and dissolution rates of needle materials, primarily composed of hyaluronic acid, for optimizing DMN-based drug delivery.

Environmental management procedures following fatal melioidosis in a captive chimpanzee (Pan troglodytes).

A 40-yr-old male captive chimpanzee (Pan troglodytes) presented with depression and anorexia for 7 days. The tentative diagnosis, following a physical examination under anesthesia, was pneumonia with sepsis. Despite antibiotic treatment and supportive care the chimpanzee died a week following presentation. Gross pathology confirmed severe purulent pneumonia and diffuse hepatosplenic abscesses. Detected in serum at the time of the initial examination, the melioidosis serum antibody titer was elevated (> 1:512). Soil samples were collected from three sites in the exhibit at three depths of 5, 15, and 30 cm. By direct and enrichment culture, positive cultures for Burkholderia pseudomallei were found at 5 and 15 cm in one site. The other two sites were positive by enrichment culture at the depth of 5 cm. To prevent disease in the remaining seven troop members, they were relocated to permit a soil treatment with calcium oxide. The exhibit remained empty for approximately 1 yr before the chimpanzees were returned. During that period, the soil in the exhibit area was again cultured as before and all samples were negative for B. pseudomallei. Following the soil treatment in the exhibit, all chimpanzees have remained free of clinical signs consistent with melioidosis.

Characterization of a bioscaffold containing polysaccharide acemannan and native collagen for pulp tissue regeneration.

Dental pulp regeneration exploits tissue engineering concepts using stem cells/scaffolds/growth-factors. Extracted collagen is commonly used as a biomaterial-scaffold due to its biocompatibility/biodegradability and mimics the natural extracellular matrix. Adding biomolecules into a collagen-scaffold enhanced pulp regeneration. Acemannan, ß-(1-4)-acetylated-polymannose, is a polysaccharide extracted from aloe vera. Acemannan is a regenerative biomaterial. Therefore, acemannan could be a biomolecule in a collagen-scaffold. Here, acemannan and native collagen were obtained and characterized. The AceCol-scaffold's physical properties were investigated using FTIR, SEM, contact angle, swelling, pore size, porosity, compressive modulus, and degradation assays. The AceCol-scaffold's biocompatibility, growth factor secretion, osteogenic protein expression, and calcification were evaluated in vitro. The AceCol-scaffolds demonstrated higher hydrophilicity, swelling, porosity, and larger pore size than the collagen scaffolds (p < 0.05). Better cell-cell and cell-scaffold adhesion, and dentin extracellular matrix protein (BSP/OPN/DSPP) expression were observed in the AceCol-scaffold, however, DSPP expression was not detected in the collagen group. Significantly increased cellular proliferation, VEGF and BMP2 expression, and mineralization were detected in the AceCol-scaffold compared with the collagen-scaffold (p < 0.05). Computer simulation revealed that acemannan's 3D structure changes to bind with collagen. In conclusion, the AceCol-scaffold synergistically provides better physical and biological properties than collagen. The AceCol-scaffold is a promising material for tissue regeneration.

Retrobulbar primitive neuroectodermal tumor in a squirrel monkey (Saimiri sciureus).

BACKGROUND: A 2.8-year-old female captive-bred common squirrel monkey (Saimiri sciureus) showed exophthalmos of the right eye, and the eye was surgically enucleated. A tumor mass was found in the eye. METHODS: Formalin-fixed tumor samples were examined histopathologically and immunohistochemically for diagnosis. RESULTS: The retrobulbar tumor mass adhered to the sclera and infiltrated the choroid. Histopathologically, tumor cells were pleomorphic, arranged in a sheet pattern, and mimicked primitive neuroectodermal cells. The tumor cells were strongly positive for precursor neuronal cell markers (beta III tubulin, neuron-specific enolase, vimentin, nestin, doublecortin, oligo2, and S-100), but negative for mature cell markers (cytokeratin, neurofilament, and glial fibrillary acidic protein) and a retinoblastoma marker (rhodopsin). CONCLUSIONS: This is the first reported case for the retrobulbar location of primitive neuroectodermal tumor in non-human primates.

The reversible effects of gossypol toxicity on male pigeons' reproductive performance.

Background and Aim: Gossypol, a cotton seed derivative, is well known for its reversible antifertility in male reproduction across species. Its antifertility and reversibility effects on male reproductive function vary among species in dose-and time-dependent manners. In this study, the antifertility potential of gossypol in pigeons was evaluated for the first time to determine whether it might be used as a dietary supplement for pigeon population control. Materials and Methods: Male pigeons were assigned into three experimental groups: The gossypol-treated group (n = 12), the sham control group (n = 6), and the negative control group (n = 6). There were two experimental periods: A gossypol-feeding period of 28 days and a gossypol-free period of 28 days. During the gossypol-feeding period, birds in the gossypol-treated group were fed 4 mg of gossypol extract per day. Birds in the sham control group were fed 0.5 mL of mixed ethanol and sunflower oil, while those in the negative control group were fed 0.5 mL of phosphate buffer saline. After the gossypol-feeding phase was completed, all remaining pigeons in all groups continued to receive their regular diet for an additional 28 days (gossypol-free phase). The body weight and semen quality of the birds in the experimental groups were compared to evaluate gossypol's antifertility effect. Results: In the gossypol-treated group as compared to the control groups, the percentages of sperm motility and viability were significantly lower at 21 days, and the percentage of normal sperm morphology was significantly lower at 28 days during the gossypol-feeding period. After gossypol withdrawal, these antifertility effects were resumed and reached a comparable semen quality to the control groups within 14 days. Conclusion: Gossypol supplementation (4 mg/day for 28 days) could lower male pigeons' reproductive performance in terms of sperm motility, viability, and sperm morphology. Such infertility was, however, reversible within 14 days after gossypol withdrawal without any side effects on the pigeons, suggesting its application as a safe contraceptive feeding for male pigeons.

Dynamics of cellular and humoral immune responses following duck Tembusu virus infection in ducks.

Duck Tembusu virus (DTMUV), an emerging avian pathogenic flavivirus, causes severe neurological disorders and acute egg drop syndrome in ducks. However, the effects of DTMUV on duck immunological components and functions remain largely unknown. In this study, the dynamics of cellular and humoral immune responses of DTMUV-infected ducks were investigated. The numbers of CD4+ and CD8+ T, B and non-T and B lymphocytes as well as the levels of neutralizing antibodies were quantified in parallel with DTMUV loads in blood and target organs. Our results demonstrated that DTMUV infection caused severe losses of non-T and B lymphocyte/myeloid cell subpopulation, and reduction in phagocytic activity during 3-5 days after infection. We also found that the numbers of T and B cells were increased during the first week of DTMUV infection. A significant negative correlation between the levels of CD4+ and CD8+ T, B and non-T and B lymphocytes and viral loads in blood and target organ (spleen) was observed during the early phase of infection. Additionally, DTMUV infection induced an early and robust neutralizing antibody response, which was associated with DTMUV-specific IgM and IgG responses. The presence of neutralizing antibody also correlated with reduction of viremia and viral load in the spleen. Overall, DTMUV elicited both cellular and humoral immune responses upon infection, in which the magnitude of these responses was correlated with reduction of viremia and viral loads in the target organ (spleen). The results suggested the critical role of both cellular and humoral immunity against DTMUV infection. This study expands our understanding of the immunological events following DTMUV infection in ducks.

Delivery and diffusion of retinal in dermis and epidermis through the combination of prodrug nanoparticles and detachable dissolvable microneedles.

To minimize chemical degradation of retinal, we graft this aldehyde on chitosan chains to make them self-assemble into pro-retinal nanoparticles (PRNs), which we then load into detachable dissolvable microneedles (DDMNs) made of 1:1 (by weight) hyaluronic acid/maltose. The presence of PRNs in the hyaluronic acid-maltose needle matrix also helps improve the microneedles' mechanical strength. Ex vivo administration of PRN-loaded DDMNs on fresh porcine ear skin shows, as observed by stereomicroscopic and confocal fluorescence microscopic analyses of the cross-sectioned tissue pieces, complete deposition followed by dissolution of the needles and diffusion of the PRNs in epidermis and dermis. Rats administered with a single dose of PRN-loaded DDMNs show significantly increased epidermal thickness as compared to rats administered with control DDMNs (no PRN). Both the PRN-loaded DDMNs and the control DDMNs produce no skin irritation in rats.

Genetic characterization of hypervirulent Klebsiella pneumoniae responsible for acute death in captive marmosets.

Klebsiella pneumoniae is a Gram-negative bacterium implicated as the causative pathogen in several medical health issues with different strains causing different pathologies including pneumonia, bloodstream infections, meningitis and infections from wounds or surgery. In this study, four captive African marmosets housed in Thailand were found dead. Necropsy and histology revealed congestion of hearts, kidneys and adrenal glands. Twenty-four bacterial isolates were obtained from these four animals with all isolates yielding identical phenotypes indicative of K. pneumoniae based on classical identification schema. All the isolates show the susceptibility to amikacin, cephalexin, doxycycline, gentamicin, and enrofloxacin with intermediate susceptibility to amoxycillin/clavulanic acid. One isolate (20P167W) was chosen for genome analysis and determined to belong to sequence type 65 (ST65). The genome of 20P167W possessed multiple virulence genes including mrk gene cluster and iro and iuc gene cluster (salmochelin and aerobactin, respectively) as well as multiple antibiotic resistance genes including bla SHV-67, bla SHV-11, oqxA, oqxB, and fosA genes resembling those found in human isolates; this isolate has a close genetic relationship with isolates from humans in Ireland, but not from Thailand and California sea lions. Phylogenetic studies using SNP show that there was no relation between genetic and geographic distributions of all known strains typing ST65, suggesting that ST65 strains may spread worldwide through multiple international transmission events rather than by local expansions in humans and/or animals. We also predict that K. pneumoniae ST65 has an ability to acquire genetic mobile element from other bacteria, which would allow Klebsiella to become an even greater public health concern.

Enhanced Nasal Deposition and Anti-Coronavirus Effect of Favipiravir-Loaded Mucoadhesive Chitosan-Alginate Nanoparticles.

Favipiravir (FVR) is a repurposed antiviral drug for treating mild to moderate cases of the novel coronavirus disease 2019 (COVID-19). However, its poor solubility and permeability limit its clinical efficacy. To overcome its physicochemical and pharmacokinetic limitations, we statistically designed a mucoadhesive chitosan-alginate nanoparticles (MCS-ALG-NPs) as a new carrier for FVR using response surface methodology, which provided suitable characteristics for transmucosal delivery. The use of mucoadhesive polymers for intranasal administration promotes the residence time and contact of FVR in the mucus membrane. The optimized FVR-MCS-ALG-NPs demonstrated superior mucoadhesion, higher permeation and deposition in the nasal mucosa, and a significant increase in the inhibition of viral replication over 35-fold compared with free FVR. The overall results suggest that MCS-ALG-NPs could be used as an effective mucoadhesive carrier to enhance the activity of FVR against COVID-19.

Detachable dissolvable microneedles: intra-epidermal and intradermal diffusion, effect on skin surface, and application in hyperpigmentation treatment.

Delivering bioactive compounds into skin tissue has long been a challenge. Using ex vivo porcine and rat skins, here we demonstrate that a detachable dissolvable microneedle (DDMN) array, a special dissolvable microneedle that allows needle detachment from the base within 2 min post administration, can effectively embed a model compound into epidermis and dermis. Diffusion of the compound from the needle embedding sites to the nearby skin tissue is demonstrated at various post administration periods. The relationship between the time that a conventional dissolvable microneedle array is left on skin without needle detachment from the base and the degree of skin surface abrasion at each microneedle penetration spot is also demonstrated on skin of human volunteers. Co-loading glutathione with vitamin C (vitC) can stabilize vitC in the DDMN. DDMN loaded with vitC and glutathione can help erasing post-acne-hyperpigmentation spots.

Evaluation of osteochondral-like tissues using human freeze-dried cancellous bone and chondrocyte sheets to treat osteochondral defects in rabbits.

Human freeze-dried cancellous bone combined with human chondrocyte sheets have recently been used to construct an osteochondral-like tissue, which resembled a cartilage layer on a subchondral bone layer. Nevertheless, the efficacy of these human tissues in a xenogeneic model has been rarely reported. Therefore, this study aimed to evaluate the potential of human freeze-dried cancellous bones combined with human chondrocyte sheets for the treatment of osteochondral defects in rabbits. The key roles of the extracellular matrix (ECM) and released cytokines in these tissues in osteochondral repair were also assessed. Triple-layered chondrocyte sheets were constructed using a temperature-responsive culture surface. Then, they were placed onto cancellous bone to form chondrocyte sheet-cancellous bone tissues. The immunostaining of collagen type II (COL2) and the proteomic analysis of the human tissues were carried out before the transplantation. In our in vitro study, the triple-layered chondrocyte sheets adhered well on the cancellous bone, and the COL2 expression was apparent throughout the tissue structures. From the proteomic analysis results, it was found that the major function of the secreted proteins found in these tissues was protein binding. The distinct pathways were focal adhesion and the ECM-receptor interaction pathways. Among the highly expressed proteins, laminin-alpha 5 (LAMA5) and fibronectin (FN) not only played roles in the protein binding and ECM-receptor interaction, but also were involved in the cytokine-mediated signaling pathway. At 12 weeks after xenogeneic transplantation, compared to the control group, the defects treated with the chondrocyte sheets showed more hyaline-like cartilage tissue, as indicated by the abundance of safranin-O and COL2 with a partial collagen type I (COL1) expression. At 4, 8, and 12 weeks, compared to the defects treated with the cancellous bone, the staining of safranin-O and COL2 was more apparent in the defects treated with the chondrocyte sheet-cancellous bone tissues. Therefore, the human chondrocyte sheets and chondrocyte sheet-cancellous bone tissues provide a potential treatment for rabbit femoral condyle defect. LAMA5 and FN found in these human xenografts and their culture media might play key roles in the ECM-receptor interaction and might be involved in the cytokine-mediated signaling pathway during tissue repair.