Generation of glial cell-derived neurotrophic factor (gdnf) morphants in zebrafish larvae by cerebroventricular microinjection of vivo morpholino.

Dopaminergic neurons in the brain are an important source of dopamine, which is a crucial neurotransmitter for wellbeing, memory, reward, and motor control. Deficiency of dopamine due to advanced age and accumulative dopaminergic neuron defects can lead to movement disorders such as Parkinson's disease. Glial cell-derived neurotrophic factor (GDNF) is one of many factors involved in dopaminergic neuron development and/or survival. However, other endogenous GDNF functions in the brain await further investigation. Zebrafish is a well-established genetic model for neurodevelopment and neurodegeneration studies. Importantly, zebrafish shares approximately 70% functional orthologs with human genes including GDNF. To gain a better understanding on the precise functional role of gdnf in dopaminergic neurons, our laboratory devised a targeted knockdown of gdnf in the zebrafish larval brain using vivo morpholino. Here, detailed protocols on the generation of gdnf morphants using vivo morpholino are outlined. This method can be applied for targeting of genes in the brain to determine specific spatiotemporal gene function in situ.

MicroRNAs-associated with FOXO3 in cellular senescence and other stress responses.

FOXO3 is a member of the FOXO transcription factor family and is known for regulating cellular survival in response to stress caused by various external and biological stimuli. FOXO3 decides cell fate by modulating cellular senescence, apoptosis and autophagy by transcriptional regulation of genes involved in DNA damage response and oxidative stress resistance. These cellular processes are tightly regulated physiologically, with FOXO3 acting as the hub that integrates signalling networks controlling them. The activity of FOXO3 is influenced by post-translational modifications, altering its subcellular localisation. In addition, FOXO3 can also be regulated directly or indirectly by microRNAs (miRNAs) or vice versa. This review discusses the involvement of various miRNAs in FOXO3-driven cellular responses such as senescence, apoptosis, autophagy, redox and inflammation defence. Given that these responses are linked and influence cell fate, a thorough understanding of the complex regulation by miRNAs would provide key information for developing therapeutic strategy and avoid unintended consequences caused by off-site targeting of FOXO3.

Exploring the perspectives of pharmaceutical experts and healthcare practitioners on senolytic drugs for vascular aging-related disorder: a qualitative study.

Objective: The field of targeting cellular senescence with drug candidates to address age-related comorbidities has witnessed a notable surge of interest and research and development. This study aimed to gather valuable insights from pharmaceutical experts and healthcare practitioners regarding the potential and challenges of translating senolytic drugs for treatment of vascular aging-related disorders. Methods: This study employed a qualitative approach by conducting in-depth interviews with healthcare practitioners and pharmaceutical experts. Participants were selected through purposeful sampling. Thematic analysis was used to identify themes from the interview transcripts. Results: A total of six individuals were interviewed, with three being pharmaceutical experts and the remaining three healthcare practitioners. The significant global burden of cardiovascular diseases presents a potentially large market size that offer an opportunity for the development and marketability of novel senolytic drugs. The pharmaceutical sector demonstrates a positive inclination towards the commercialization of new senolytic drugs targeting vascular aging-related disorders. However potential important concerns have been raised, and these include increasing specificity toward senescent cells to prevent off-site targeting, thus ensuring the safety and efficacy of these drugs. In addition, novel senolytic therapy for vascular aging-related disorders may encounter competition from existing drugs that treat or manage risk factors of cardiovascular diseases. Healthcare practitioners are also in favor of recommending the novel senolytic drugs for vascular aging-related disorders but cautioned that its high cost may hinder its acceptance among patients. Besides sharing the same outcome-related concerns as with the pharmaceutical experts, healthcare practitioners anticipated a lack of awareness among the general public regarding the concept of targeting cellular senescence to delay vascular aging-related disorders, and this knowledge gap extends to healthcare practitioner themselves as well. Conclusion: Senolytic therapy for vascular aging-related disorders holds great promise, provided that crucial concerns surrounding its outcomes and commercial hurdles are effectively addressed.

Quassinoids from Eurycoma longifolia Jack roots and their potential inhibitory activity against human benign prostatic hyperplasia cells (BPH-1) and testosterone-induced BPH rat model.

Benign prostate hyperplasia (BPH) is an enlargement of the prostate gland, because of hormonal changes in aging males which contribute significantly to excessive proliferation over apoptosis of prostatic cells. The anti-proliferative and induced apoptotic activities of Eurycoma longifolia quassinoids on cancer cell lines could be promising therapeutic targets on BPH. Hitherto, no report of the quassinoids against BPH problem was available. In this study, a systematic phytochemical fractionation of the root extract, TAF2 was performed, which led to the discovery of nine previously described C20 quassinoids (1-9). Two undescribed C20 (10 and 12) and one undescribed (11) C19 quassinoids were identified by detailed NMR and HR-ESI-MS data analysis. Their absolute configurations were assigned by ECD spectral analysis. The quassinoids (1-12) were tested for inhibitory activity against the proliferation of human BPH-1 and human skin Hs27 fibroblast cells cultured in vitro. 1, 2 and 3 at 10 µM significantly reduced BPH-1 cell viability and were cytotoxic to Hs27 fibroblast cells. 2 was selected for further study of anti-BPH activity against testosterone induced BPH rats. At 5 mg/kg, 2 reduced the rat prostatic weight and prostatic index, consistent with the decrease in papillary acini number and epithelial thickness of the prostate tissues. These quassinoids may be potential anti-BPH compounds that require further studies.

The clinical utility of CD163 in viral diseases.

Macrophage activation and hypercytokinemia are notable presentations in certain viral infections leading to severe disease and poor prognosis. Viral infections can cause macrophage polarization into the pro-inflammatory M1 or anti-inflammatory M2 phenotype. Activated M1 macrophages usually restrict viral replication whereas activated M2 macrophages suppress inflammation and promote tissue repair. In response to inflammatory stimuli, macrophages polarize to the M2 phenotype expressing hemoglobin scavenger CD163 surface receptor. The CD163 receptor is shed as the soluble form, sCD163, into plasma or tissue fluids. sCD163 causes detoxification of pro-oxidative hemoglobin which produces anti-inflammatory metabolites that promote the resolution of inflammation. Hence, increased CD163 expression in tissues and elevated circulatory levels of sCD163 have been associated with acute and chronic inflammatory diseases. CD163 and other macrophage activation markers have been commonly included in the investigation of disease pathogenesis and progression. This review provides an overview of the involvement of CD163 in viral diseases. The clinical utility of CD163 in viral disease diagnosis, progression, prognosis and treatment evaluation is discussed.

Senotherapeutics for mesenchymal stem cell senescence and rejuvenation.

Mesenchymal stem cells (MSCs) are susceptible to replicative senescence and senescence-associated functional decline, which hampers their use in regenerative medicine. Senotherapeutics are drugs that target cellular senescence through senolytic and senomorphic functions to induce apoptosis and suppress chronic inflammation caused by the senescence-associated secreted phenotype (SASP), respectively. Therefore, senotherapeutics could delay aging-associated degeneration. They could also be used to eliminate senescent MSCs during in vitro expansion or bioprocessing for transplantation. In this review, we discuss the role of senotherapeutics in MSC senescence, rejuvenation, and transplantation, with examples of some tested compounds in vitro. The prospects, challenges, and the way forward in clinical applications of senotherapeutics in cell-based therapeutics are also discussed.

Hypoxia-regulated microRNAs: the molecular drivers of tumor progression.

Hypoxia is a common feature of the tumor microenvironment (TME) of nearly all solid tumors, leading to therapeutic failure. The changes in stiffness of the extracellular matrix (ECM), pH gradients, and chemical balance that contribute to multiple cancer hallmarks are closely regulated by intratumoral oxygen tension via its primary mediators, hypoxia-inducible factors (HIFs). HIFs, especially HIF-1α, influence these changes in the TME by regulating vital cancer-associated signaling pathways and cellular processes including MAPK/ERK, NF-κB, STAT3, PI3K/Akt, Wnt, p53, and glycolysis. Interestingly, research has revealed the involvement of epigenetic regulation by hypoxia-regulated microRNAs (HRMs) of downstream target genes involved in these signaling. Through literature search and analysis, we identified 48 HRMs that have a functional role in the regulation of 5 key cellular processes: proliferation, metabolism, survival, invasion and migration, and immunoregulation in various cancers in hypoxic condition. Among these HRMs, 17 were identified to be directly associated with HIFs which include miR-135b, miR-145, miR-155, miR-181a, miR-182, miR-210, miR-224, miR-301a, and miR-675-5p as oncomiRNAs, and miR-100-5p, miR-138, miR-138-5p, miR-153, miR-22, miR-338-3p, miR-519d-3p, and miR-548an as tumor suppressor miRNAs. These HRMs serve as a potential lead in the development of miRNA-based targeted therapy for advanced solid tumors. Future development of combined HIF-targeted and miRNA-targeted therapy is possible, which requires comprehensive profiling of HIFs-HRMs regulatory network, and improved formula of the delivery vehicles to enhance the therapeutic kinetics of the targeted cancer therapy (TCT) moving forward.

Cartilage debris and osteoarthritis risk factors influence gene expression in the synovium in end stage osteoarthritis.

BACKGROUND: Gene expression in healthy synovium remains poorly characterised. Thus, synovial functional activity changes associated with osteoarthritis (OA) are difficult to define. This study sought to identify differentially expressed genes (DEG) of end-stage OA and assess the influence of OA risk factors on these DEG. METHODS: Anonymised patient clinical data and x-ray images were analysed. Osteoarthritic and non-osteoarthritic patients with soft tissue or traumatic knee injuries were matched for body mass index (BMI) and sex. Tissue samples were partitioned for immunocytochemistry (IHC) and microarray analysis. Multiple bioinformatics applications were utilised to determine changes in functional and canonical pathway activation. RESULTS: Age, disease-modifying injections and hypertension were confounding factors between patient groups. Inflammation was present in all tissues. Cartilage debris and inflammatory aggregates were noted in many osteoarthritic patient tissues. IHC and expression analyses revealed upregulation of synoviolin 1 (SYVN1) in osteoarthritic synovium. Significant differential expression was noted in 2084 genes. Osteoarthritic synovium displayed a significant upregulation of 95% of DEG coding for proteins, relative to non-osteoarthritic synovium tissues. Unfolded protein response (UPR)-related genes were upregulated in osteoarthritic synovium; gene expression of molecules within many canonical pathways including protein ubiquitination and UPR pathways was modified by BMI and sex. CONCLUSIONS: The synovium of all three pathologies exhibited elements of an inflammatory response. Cartilage debris, age, BMI and sex influence DEG of osteoarthritic synovium. UPR pathway is the top deregulated canonical pathway identified in osteoarthritic synovium regardless of BMI and sex, while typical OA-associated inflammatory and matrix gene responses were minimal.

Correction: Alpha-Tomatine Attenuation of In Vivo Growth of Subcutaneous and Orthotopic Xenograft Tumors of Human Prostate Carcinoma PC-3 Cells Is Accompanied by Inactivation of Nuclear Factor-Kappa B Signaling.

[This corrects the article DOI: 10.1371/journal.pone.0057708.].

CircRNA-miRNA interactions in atherogenesis.

Atherosclerosis is the major cause of coronary artery disease (CAD) which includes unstable angina, myocardial infarction, and heart failure. The onset of atherogenesis, a process of atherosclerotic lesion formation in the intima of arteries, is driven by lipid accumulation, a vicious cycle of reactive oxygen species (ROS)-induced oxidative stress and inflammatory reactions leading to endothelial cell (EC) dysfunction, vascular smooth muscle cell (VSMC) activation, and foam cell formation which further fuel plaque formation and destabilization. In recent years, there is a surge in the number of publications reporting the involvement of circular RNAs (circRNAs) in the pathogenesis of cardiovascular diseases, cancers, and metabolic syndromes. These studies have advanced our understanding on the biological functions of circRNAs. One of the most common mechanism of action of circRNAs reported is the sponging of microRNAs (miRNAs) by binding to the miRNAs response element (MRE), thereby indirectly increases the transcription of their target messenger RNAs (mRNAs). Individual networks of circRNA-miRNA-mRNA associated with atherogenesis have been extensively reported, however, there is a need to connect these findings for a complete overview. This review aims to provide an update on atherogenesis-related circRNAs and analyze the circRNA-miRNA-mRNA interactions in atherogenesis. The atherogenic mechanisms and clinical relevance of each atherogenesis-related circRNA were systematically discussed for better understanding of the knowledge gap in this area.

Histopathological dataset and demographic details of synovial tissues from patients with end-stage osteoarthritis, soft tissue and traumatic injuries of the knee.

Degradation of articular cartilage is the defining feature of end-stage osteoarthritis (OA) with osteophytes, subchondral sclerosis, malalignment and joint space narrowing being additional indicators of advanced disease. Obesity, older age and female gender are OA risk factors. Differing degrees of synovitis are observed in OA, soft tissue and traumatic injuries of the knee. The synovium is also subject to systemic, enhanced lipids and inflammatory mediators characteristic of obesity. Synovial cellular composition changes specific to OA and associated with its handling of cartilage debris are unclear. Triangulation of data from three knee pathologies was used to highlight findings pertaining to OA compared to non-OA. OA patient data was compared to non-OA from knee ligament and tibial frature patients at surgery. Knee pathology, gender and BMI informed patient identification. Once consented, patient inclusion and characterisation utilised data from clinical assessments, blood tests, function scores, and radiological imaging, scores and intraoperative assessment. Intra-operative synovial tissues from the same site and processed identically underpins in-depth analyses and comparisons of histopathological images from these different knee pathologies. This supports the identification of distinct changes in the cellular composition of the knee synovium characteristic of OA. This data underpins a better understanding of OA pathogenesis and disease progression vital for the design of targeted therapeutics. The tissue and cell data include detailed results from the semi-quantitative synovitis score established by Krenn and observational data for morphological features such as cartilage debris inclusion, inflammatory cells aggregate and infiltration. This histopathological data is presented in the context of detailed clinical and functional information. This data and the holistic study design can be used as a foundation for the multifactorial collection and analysis of clinical data from OA patients, OA severity measures, tissue immuno-histology and synovial inflammation analysis to underpin the details and comparisons needed in further studies into OA and its treatment globally.

Naturally-derived endoplasmic reticulum stress inhibitors for osteoarthritis?

Osteoarthritis (OA) is a chronic degenerative disease that affects the whole synovial joint. OA causes severe pain and disability that significantly affects the livelihood of an individual and incurs a huge socioeconomic burden. Current management strategies are limited to supporting functional improvement with physiotherapy and pain reduction as there are no drugs available that can reverse the progression of OA with only joint replacement surgery for late stage OA. OA is associated with advancing age and obesity, both of which compromise the functions of key endoplasmic reticulum (ER) molecular chaperones leading to improper protein folding and ER stress. Failure to restore protein homeostasis leads to increased cellular stress and eventually apoptotic cell death. Cartilage is avascular and is dependent on its constituent cells, chondrocytes, for extracellular matrix maintenance. Chondrocytes have limited proliferative capacity and their apoptosis eventually leads to extracellular matrix loss and cartilage degeneration. Recent studies on attenuating ER stress and chondrocytes apoptosis offer a credible strategy for reducing OA progression. The established roles of ER stress responses in OA have paved the way for targeted drug discovery studies aiming to mitigate ER stress and OA progression. In this review, in vitro, pre-clinical and clinical evidence of naturally-derived ER stress inhibitors for OA, the prospect and challenges in bringing these compounds to clinics are discussed.

MiR-107 inhibits the sprouting of intersegmental vessels of zebrafish embryos.

MicroRNAs (miRNAs) play important roles in various biological processes. Our previous study showed that inhibition of MTOR with rapamycin treatment suppressed human endothelial cell tube formation, concomitant with the down-regulation of miR-107. Similarly, inhibition of Ztor by rapamycin also suppressed vascular development in zebrafish embryos. To gain a better understanding of the role of miR-107 and MTOR in vascular development, the present study sought to validate its function by over-expressing miR-107 in zebrafish embryos via microinjection with mimic miR-107 duplexes. Alkaline phosphatase (ALP) staining was used to visualise blood vessels in the zebrafish embryo, and expressions of Pten, Ztor and Rap1 were investigated by immunoblotting. Over-expression of miR-107 in zebrafish embryos inhibited the sprouting of intersegmental vessels (ISVs) with concomitant down-regulation of phosphorylated Rps6 expression, which confirmed the inhibition of Ztor signalling. As expected, pten, which is the target of miR-107, was down-regulated. Interestingly, Rap1, a small GTPase protein that is involved in intersomitic vessels sprouting during angiogenesis, was also down-regulated when miR-107 was over-expressed. Overall, our findings suggest that miR-107 and Ztor-mediated suppression of vascular development in zebrafish embryo involves Rap1.

A 3D Microfluidic ELISA for the Detection of Severe Dengue: Sensitivity Improvement and Vroman Effect Amelioration by EDC-NHS Surface Modification.

Serum is commonly used as a specimen in immunoassays but the presence of heterophilic antibodies can potentially interfere with the test results. Previously, we have developed a microfluidic device called: 3D Stack for enzyme-linked immunosorbent assay (ELISA). However, its evaluation was limited to detection from a single protein solution. Here, we investigated the sensitivity of the 3D Stack in detecting a severe dengue biomarker-soluble CD163 (sCD163)-within the serum matrix. To determine potential interactions with serum matrix, a spike-and-recovery assay was performed, using 3D Stacks with and without surface modification by an EDC-NHS (N-ethyl-N'-(3-(dimethylamino)propyl)carbodiimide/N-hydroxysuccinimide) coupling. Without surface modification, a reduced analyte recovery in proportion to serum concentration was observed because of the Vroman effect, which resulted in competitive displacement of coated capture antibodies by serum proteins with stronger binding affinities. However, EDC-NHS coupling prevented antibody desorption and improved the sensitivity. Subsequent comparison of sCD163 detection using a 3D Stack with EDC-NHS coupling and conventional ELISA in dengue patients' sera revealed a high correlation (R = 0.9298, p < 0.0001) between the two detection platforms. Bland-Altman analysis further revealed insignificant systematic error between the mean differences of the two methods. These data suggest the potentials of the 3D Stack for further development as a detection platform.

Adipogenesis and therapeutic potentials of antiobesogenic phytochemicals: Insights from preclinical studies.

Obesity is one of the most serious public health problems in both developed and developing countries in recent years. While lifestyle and diet modifications are the most important management strategies of obesity, these may be insufficient to ensure long-term weight reduction in certain individuals and alternative strategies including pharmacotherapy need to be considered. However, drugs option remains limited due to low efficacy and adverse effects associated with their use. Hence, identification of safe and effective alternative therapeutic agents remains warranted to combat obesity. In recent years, bioactive phytochemicals are considered as valuable sources for the discovery of new pharmacological agents for the treatment of obesity. Adipocyte hypertrophy and hyperplasia increases with obesity and undergo molecular and cellular alterations that can affect systemic metabolism giving rise to metabolic syndrome and comorbidities such as type 2 diabetes and cardiovascular diseases. Many phytochemicals have been reported to target adipocytes by inhibiting adipogenesis, inducing lipolysis, suppressing the differentiation of preadipocytes to mature adipocytes, reducing energy intake, and boosting energy expenditure mainly in vitro and in animal studies. Nevertheless, further high-quality studies are needed to firmly establish the clinical efficacy of these phytochemicals. This review outlines common pathways involved in adipogenesis and phytochemicals targeting effector molecules of these pathways, the challenges faced and the way forward for the development of phytochemicals as antiobesity agents.

Isolation of Streptococcus cuniculi from corneal lesion in laboratory-raised mice.

Corneal lesions appearing as white mass beneath intact epithelium, with ocular discharge in one mouse, was observed in a batch of laboratory-raised BALB/c mice (n=9 of 56). The affected mice remained active, well-groomed and had normal appetite. Isolates recovered from swab cultures of the external and internal contents of the eye had partial 16S rRNA gene sequence of 99.1% similarity to Streptococcus cuniculi. No previous report of S. cuniculi infection in laboratory rodents has been presented. The isolate was susceptible to all antibiotics tested. We suggest S. cuniculi is an opportunistic bacteria in laboratory mice but are uncertain of its source. Our findings revealed that S. cuniculi is able to colonize laboratory mice and should be considered when mice present with eye lesion or ocular discharge.

Modulation of the mechanical responses of synovial fibroblasts by osteoarthritis-associated inflammatory stressors.

OBJECTIVES: To compare mechanobiological response of synovial fibroblasts (SFb) from OA patient cohorts under mechanical load and inflammatory stressors for better understanding of SFb homeostatic functions. METHODS: Primary SFb isolated from knee synovium of OA obese (OA-ob:SFb), OA-pre-obese (OA-Pob:SFb), non-OA arthroscopic (scope:SFb), and non-OA arthroscopic with cartilage damage (scope-CD:SFb) were exposed to OA-conditioned media (OACM), derived from OA obese (OA-ob:CM), OA-pre-obese (OA-Pob:CM), and mechanical stretch at either 0 %, 6 % or 10 % for 24 h. Differences in the mRNA levels of genes involved in extracellular matrix production, inflammation and secretory activity were measured. RESULTS: Despite the significant BMI differences between the OA-ob and OA-Pob groups, OA-Pob has more patients with underlying dyslipidaemia, and low-grade synovitis with higher levels of secreted proteins, CXCL8, COL4A1, CCL4, SPARC and FGF2 in OA-Pob:CM. All primary SFb exhibited anti-proliferative activity with both OA-CM. Mechanical stretch stimulated lubricin production in scope:SFb, higher TGFß1 and COL1A1 expressions in scope-CD:SFb. OA-Pob:CM stimulated greater detrimental effects than the OA-ob:CM, with higher pro-inflammatory cytokines, IL1ß, IL6, COX2 and proteases such as aggrecanases, ADAMTS4 and ADAMTS5, and lower ECM matrix, COL1A1 expressions in all SFb. OA-ob:SFb were unresponsive but expressed higher pro-inflammatory cytokines under OA-Pob:CM treatment. CONCLUSION: Both mechanical and inflammatory stressors regulate SFb molecular functions with heterogeneity in responses that are dependent on their pathological tissue of origins. While mechanical stretch promotes a favorable effect with enhanced lubricin production in scope:SFb and TGFß1 and COL1A1 in scope-CD:SFb, the presence of excessively high OA-associated inflammatory mediators in OA-Pob:CM, predominantly SPARC, CXCL8 and FGF2 drive all SFb regardless of pathology, towards greater pro-inflammatory activities.

The use of the health belief model to assess predictors of intent to receive the COVID-19 vaccine and willingness to pay.

BACKGROUND: The development of a vaccine against SARS-CoV-2 infection is on the way. To prepare for public availability, the acceptability of a hypothetical COVID-19 vaccine and willingness to pay (WTP) were assessed to provide insights into future demand forecasts and pricing considerations. METHODS: A cross-sectional survey was conducted from 3 to 12 April 2020. The health belief model (HBM) was used to assess predictors of the intent to receive the vaccine and the WTP. RESULTS: A total of 1,159 complete responses was received. The majority reported a definite intent to receive the vaccine (48.2%), followed by a probable intent (29.8%) and a possible intent (16.3%). Both items under the perceived benefits construct in the HBM, namely believe the vaccination decreases the chance of infection (OR = 2.51, 95% CI 1.19-5.26) and the vaccination makes them feel less worry (OR = 2.19, 95% CI 1.03-4.65), were found to have the highest significant odds of a definite intention to take the vaccine. The mean ± standard deviation (SD) for the amount that participants were willing to pay for a dose of COVID-19 vaccine was MYR$134.0 (SD±79.2) [US$30.66 ± 18.12]. Most of the participants were willing to pay an amount of MYR$100 [US$23] (28.9%) and MYR$50 [US$11.5] (27.2%) for the vaccine. The higher marginal WTP for the vaccine was influenced by no affordability barriers as well as by socio-economic factors, such as higher education levels, professional and managerial occupations and higher incomes. CONCLUSIONS: The findings demonstrate the utility of HBM constructs in understanding COVID-19 vaccination intention and WTP.

Multidimensional social and cultural norms influencing HPV vaccine hesitancy in Asia.

HPV vaccine hesitancy in Asia is unique compared to Western countries due to multidimensional social-cultural norms that influence beliefs regarding vaccination. Reviews on HPV vaccine hesitancy in Asia lack of in-depth discussion regarding the traditional and social-cultural norms dimensions. This paper puts forward opinions in which culture, normative beliefs, and religion influence HPV vaccine hesitancy in Asian countries. Issues surrounding HPV hesitancy among parents, young adult women, adult women, men and the sexual and gender minority people in Asian countries were highlighted. The shortage of HPV vaccine supply would soon be reduced as some Asian countries are on the way to producing the HPV vaccine which production is currently dominated by Western European countries. The culture of favoring imported Western products among many in Asia and in addition to long-existing fake vaccine crisis pose a challenge for the newly emerging HPV vaccine produced in Asia. Some recommendations, research gaps, and future research needs were discussed.