Development of a luciferase-based Gram-positive bacterial reporter system for the characterization of antimicrobial agents.

Mechanistic investigations are of paramount importance in elucidating the modes of action of antibiotics and facilitating the discovery of novel drugs. We reported a luciferase-based reporter system using bacterial cells to unveil mechanisms of antimicrobials targeting transcription and translation. The reporter gene Nluc encoding NanoLuciferase (NanoLuc) was integrated into the genome of the Gram-positive model organism, Bacillus subtilis, to generate a reporter strain BS2019. Cellular transcription and translation levels were assessed by quantifying the amount of Nluc mRNA as well as the luminescence catalyzed by the enzyme NanoLuc. We validated this system using three known inhibitors of transcription (rifampicin), translation (chloramphenicol), and cell wall synthesis (ampicillin). The B. subtilis reporter strain BS2019 successfully revealed a decline in Nluc expression by rifampicin and NanoLuc enzyme activity by chloramphenicol, while ampicillin produced no observable effect. The assay was employed to characterize a previously discovered bacterial transcription inhibitor, CUHK242, with known antimicrobial activity against drug-resistant Staphylococcus aureus. Production of Nluc mRNA in our reporter BS2019 was suppressed in the presence of CUHK242, demonstrating the usefulness of the construct, which provides a simple way to study the mechanism of potential antibiotic candidates at early stages of drug discovery. The reporter system can also be modified by adopting different promoters and reporter genes to extend its scope of contribution to other fields of work. IMPORTANCE: Discovering new classes of antibiotics is desperately needed to combat the emergence of multidrug-resistant pathogens. To facilitate the drug discovery process, a simple cell-based assay for mechanistic studies is essential to characterize antimicrobial candidates. In this work, we developed a luciferase-based reporter system to quantify the transcriptional and translational effects of potential compounds and validated our system using two currently marketed drugs. Reporter strains generated in this study provide readily available means for identifying bacterial transcription inhibitors as prospective novel antibacterials. We also provided a series of plasmids for characterizing promoters under various conditions such as stress.

Fabrication of Electronically Conductive Protein-Heme Nanowires for Power Harvesting.

Electronically conductive protein-based materials can enable the creation of bioelectronic components and devices from sustainable and nontoxic materials, while also being well-suited to interface with biological systems, such as living cells, for biosensor applications. However, as proteins are generally electrical insulators, the ability to render protein assemblies electroactive in a tailorable manner can usher in a plethora of useful materials. Here, an approach to fabricate electronically conductive protein nanowires is presented by aligning heme molecules in proximity along protein filaments, with these nanowires also possessing charge transfer abilities that enable energy harvesting from ambient humidity. The heme-incorporated protein nanowires demonstrate electron transfer over micrometer distances, with conductive atomic force microscopy showing individual nanowires having comparable conductance to other previously characterized heme-based bacterial nanowires. Exposure of multilayer nanowire films to humidity produces an electrical current, presumably through water molecules ionizing carboxyl groups in the filament and creating an unbalanced total charge distribution that is enhanced by the heme. Incorporation of heme and potentially other metal-center porphyrin molecules into protein nanostructures could pave the way for structurally- and electrically-defined protein-based bioelectronic devices.

Streptococcus anginosus promotes gastric inflammation, atrophy, and tumorigenesis in mice.

Streptococcus anginosus (S. anginosus) was enriched in the gastric mucosa of patients with gastric cancer (GC). Here, we show that S. anginosus colonized the mouse stomach and induced acute gastritis. S. anginosus infection spontaneously induced progressive chronic gastritis, parietal cell atrophy, mucinous metaplasia, and dysplasia in conventional mice, and the findings were confirmed in germ-free mice. In addition, S. anginosus accelerated GC progression in carcinogen-induced gastric tumorigenesis and YTN16 GC cell allografts. Consistently, S. anginosus disrupted gastric barrier function, promoted cell proliferation, and inhibited apoptosis. Mechanistically, we identified an S. anginosus surface protein, TMPC, that interacts with Annexin A2 (ANXA2) receptor on gastric epithelial cells. Interaction of TMPC with ANXA2 mediated attachment and colonization of S. anginosus and induced mitogen-activated protein kinase (MAPK) activation. ANXA2 knockout abrogated the induction of MAPK by S. anginosus. Thus, this study reveals S. anginosus as a pathogen that promotes gastric tumorigenesis via direct interactions with gastric epithelial cells in the TMPC-ANXA2-MAPK axis.

Harnessing the Structural and Functional Diversity of Protein Filaments as Biomaterial Scaffolds.

The natural ability of many proteins to polymerize into highly structured filaments has been harnessed as scaffolds to align functional molecules in a diverse range of biomaterials. Protein-engineering methodologies also enable the structural and physical properties of filaments to be tailored for specific biomaterial applications through genetic engineering or filaments built from the ground up using advances in the computational prediction of protein folding and assembly. Using these approaches, protein filament-based biomaterials have been engineered to accelerate enzymatic catalysis, provide routes for the biomineralization of inorganic materials, facilitate energy production and transfer, and provide support for mammalian cells for tissue engineering. In this review, we describe how the unique structural and functional diversity in natural and computationally designed protein filaments can be harnessed in biomaterials. In addition, we detail applications of these protein assemblies as material scaffolds with a particular emphasis on applications that exploit unique properties of specific filaments. Through the diversity of protein filaments, the biomaterial engineer's toolbox contains many modular protein filaments that will likely be incorporated as the main structural component of future biomaterials.

Synthesis and biological evaluation of nusbiarylin derivatives as bacterial rRNA synthesis inhibitor with potent antimicrobial activity against MRSA and VRSA.

Bacterial transcription is a valid but underutilized target for antimicrobial agent discovery because of its function of bacterial RNA synthesis. Bacterial transcription factors NusB and NusE form a transcription complex with RNA polymerase for bacterial ribosomal RNA synthesis. We previously identified a series of diarylimine and -amine inhibitors capable of inhibiting the interaction between NusB and NusE and exhibiting good antimicrobial activity. To further explore the structural viability of these inhibitors, coined "nusbiarylins", 36 new derivatives containing diverse substituents at the left benzene ring of inhibitors were synthesized based upon isosteric replacement and the structure-activity relationship concluded from earlier studies. Some of the derivatives displayed good to excellent antibacterial efficacy towards a panel of clinically significant pathogens including methicillin-resistance Staphylococcus aureus (MRSA) and vancomycin-resistance S. aureus (VRSA). In particular, compound 22r exhibited the best antimicrobial activity with a minimum inhibitory concentration (MIC) of 0.5 µg/mL. Diverse mechanistic studies validated the capability of 22r inhibiting the function of NusB protein and bacterial rRNA synthesis. In silico study of drug-like properties also provided promising results. Overall, this series of derivatives showed potential antimicrobial activity and drug-likeness and provided guidance for further optimization.

Exploring the impact of physicochemical properties of liposomal formulations on their in vivo fate.

Liposomes, vesicles composed of a phospholipid bilayer, are considered a remarkably advanced drug delivery system due to their unique properties, including their biocompatibility and biodegradability, and their capability to reduce toxicity of encapsulated drugs. The in vivo fate of an encapsulated drug in the form of liposome depends on both the drug and the liposome characteristics and the patient pathophysiology. In this review, the impact of the physicochemical properties of liposomes (lipid composition, size and charge) on their pharmacokinetics (systemic absorption, distribution and clearance) was discussed. In the rest, a comprehensive overview of different mechanisms of liposomal uptake by the cells (fusion, lipid transfer, and endocytosis) was provided. The importance of lipid composition and size of liposomes, cell type, and protein corona for each uptake pathway was explained.

Effectiveness of bio-dispersant in homogenizing hydroxyapatite for proliferation and differentiation of osteoblast.

Hydroxyapatite (HA), an inorganic compound, plays an essential role in the proliferation and differentiation of bone cells. Using cellulose nanocrystals (CNCs) as green dispersants to improve homogenization of HA is promising in the fabrication of nanocomposite scaffolds with biocompatibility for bone tissue engineering. The HA/CNC (HC) nanoparticle suspension was incorporated in polyvinyl alcohol (PVA)-based scaffold to investigate the physical and chemical properties. The PVA/HC composites demonstrated high porous structure and swelling ability for cell attachment and a 3-fold improvement in compressive modulus compared with free HC scaffold. Moreover, the presence of HC nanoparticles has promoted the proliferation and mineralization of pre-osteoblast. Our findings could provide an effective strategy by using bio-dispersants to incorporate mineral elements into synthetic polymers for the fabrication of functional tissue engineering scaffolds.

Evaluation of carbon sources from sugar industry to bacterial nanocellulose produced by Komagataeibacter xylinus.

Nanocellulose derived from microorganism is crucial bio-based products due to its unique physicochemical and mechanical properties for material science. Thus, optimizing bacterial cellulose (BNC) production is essential to widen applications and reduce production cost. Using various carbon sources derive from fruits as alternatives for synthesizing BNC could produce a low-cost BNC with comparable properties. Although Komagataeibacter xylinus grown in different natural juices, including clarified juice (CJ), sugarcane juice (SC) and coconut juice (CN) demonstrated a lower yield than that of control medium (HS), FTIR confirmed no change in chemical functional groups of BNCs. Similarly, different sugar sources have slightly effects on mechanical and thermal properties of BNC. However, the internal morphology illustrated the pore structure in oval shape for HS and CN while CJ and SC resulted in irregular pores which could lead to the highest crystallinity index value for BNC from HS compared to that from alternative media.

The first case study of wastewater-based epidemiology of COVID-19 in Hong Kong.

Early detection and surveillance of severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) virus are key pre-requisites for the effective control of coronavirus disease (COVID-19). So far, sewage testing has been increasingly employed as an alternative surveillance tool for this disease. However, sampling site characteristics impact the testing results and should be addressed in the early use stage of this emerging tool. In this study, we implemented the sewage testing for SARS-CoV-2 virus across sampling sites with different sewage system characteristics. We first validated a testing method using "positive" samples from a hospital treating COVID-19 patients. This method was used to test 107 sewage samples collected during the third wave of the COVID-19 outbreak in Hong Kong (from June 8 to September 29, 2020), covering sampling sites associated with a COVID-19 hospital, public housing estates, and conventional sewage treatment facilities. The highest viral titer of 1975 copy/mL in sewage was observed in a sample collected from the isolation ward of the COVID-19 hospital. Sewage sampling at individual buildings detected the virus 2 days before the first cases were identified. Sequencing of the detected viral fragment confirmed an identical nucleotide sequence to that of the SARS-CoV-2 isolated from human samples. The virus was also detected in sewage treatment facilities, which serve populations of approximately 40,000 to more than one million people.

Structural Determination of a Filamentous Chaperone to Fabricate Electronically Conductive Metalloprotein Nanowires.

The transfer of electrons through protein complexes is central to cellular respiration. Exploiting proteins for charge transfer in a controllable fashion has the potential to revolutionize the integration of biological systems and electronic devices. Here we characterize the structure of an ultrastable protein filament and engineer the filament subunits to create electronically conductive nanowires under aqueous conditions. Cryoelectron microscopy was used to resolve the helical structure of gamma-prefoldin, a filamentous protein from a hyperthermophilic archaeon. Conjugation of tetra-heme c3-type cytochromes along the longitudinal axis of the filament created nanowires capable of long-range electron transfer. Electrochemical transport measurements indicated networks of the nanowires capable of conducting current between electrodes at the redox potential of the cytochromes. Functionalization of these highly engineerable nanowires with other molecules, such as redox enzymes, may be useful for bioelectronic applications.

Loss of Bcl-G, a Bcl-2 family member, augments the development of inflammation-associated colorectal cancer.

Gastrointestinal epithelial cells provide a selective barrier that segregates the host immune system from luminal microorganisms, thereby contributing directly to the regulation of homeostasis. We have shown that from early embryonic development Bcl-G, a Bcl-2 protein family member with unknown function, was highly expressed in gastrointestinal epithelial cells. While Bcl-G was dispensable for normal growth and development in mice, the loss of Bcl-G resulted in accelerated progression of colitis-associated cancer. A label-free quantitative proteomics approach revealed that Bcl-G may contribute to the stability of a mucin network, which when disrupted, is linked to colon tumorigenesis. Consistent with this, we observed a significant reduction in Bcl-G expression in human colorectal tumors. Our study identifies an unappreciated role for Bcl-G in colon cancer.

Influenza A Virus Infections in Dromedary Camels, Nigeria and Ethiopia, 2015-2017.

We examined nasal swabs and serum samples acquired from dromedary camels in Nigeria and Ethiopia during 2015-2017 for evidence of influenza virus infection. We detected antibodies against influenza A(H1N1) and A(H3N2) viruses and isolated an influenza A(H1N1)pdm09-like virus from a camel in Nigeria. Influenza surveillance in dromedary camels is needed.

Production of hydroxyapatite-bacterial nanocellulose scaffold with assist of cellulose nanocrystals.

Bacterial nanocellulose/hydroxyapatite/cellulose nanocrystal (BHC) composites were synthesized via in-situ synthesis using cellulose nanocrystals (CNCs) to improve colloidal stability and the dispersion of hydroxyapatite (HA) during the bacterial nanocellulose (BNC) cultivation period. Transmission electron microscopy images and energy dispersive spectroscopy (EDS) results confirmed the dispersion of HA on the CNC particles with a Ca/P ratio of 1.66 corresponding to that of the stoichiometric HA. The SEM images and EDS results showed that the integration of the HA and BNC network without CNC assistance (BHA (0.25 and 0.5 wt.%) composites) was less than that for BHC at both concentrations. Fourier-transform infrared analysis, XRD and thermal degradation revealed the effect of HA on the BHC composites with a decreased CrI% and improved thermal property. Cytotoxicity proved the potential for using BHC composites for bone tissue engineering scaffold with cell viability up to 83.4 ± 3.6% compared to the negative control (99.2 ± 0.08%).

Homogenous isolation of individualized bacterial nanofibrillated cellulose by high pressure homogenization.

Varying levels of high pressure homogenization (HPH) were applied to disintegrate bacterial nanofibrillated cellulose (BNFC) from bacterial cellulose (BC). HPH was considered as a simple, non-toxic and highly efficient physical method for nanofibrillated cellulose extraction. The blended BC passed through chambers at high pressures of 68, 138 and 207MPa for 30 cycles. The particle size confirmed disintegration of the BC network fibers to bundles of BNFC and the atomic force microscopy images showed the decreased diameter of individual BNFC in the range 36-67nm. Fourier transform infrared spectroscopy measurement indicated there were no change in the chemical functional groups of the BNFC compared with BC. The decreased crystallinity index and crystallite size of BNFC with increased pressure confirmed the effect of HPH on the BNFC. Nevertheless, BNFC at 207MPa had the lowest thermal stability due to having the highest surface area, which resulted in the minimum nanofiber diameter.

Skeletal characterization of an osteoblast-specific vitamin D receptor transgenic (ObVDR-B6) mouse model.

BACKGROUND: Overexpression of the human vitamin D receptor (hVDR) transgene under control of the human osteocalcin promoter in FVB/N mice (OSVDR) was previously demonstrated to exhibit increased cortical and trabecular bone volume and strength due to decreased bone resorption and increased bone formation. An important question to address is whether the OSVDR bone phenotype persists on an alternative genetic background such as C57Bl6/J. METHODS: OSVDR mice (OSV3 line) were backcrossed onto the C57Bl6/J genetic background for at least 6 generations to produce OSVDR mice with 98.4% C57Bl6/J congenicity (ObVDR-B6 mice). Hemizygous male and female ObVDR-B6 and littermate wild-type (WT) mice were fed a standard laboratory chow diet and killed at 3, 9 and 20 weeks of age for analyses of biochemical and structural variables and dynamic indices of bone histomorphometry. RESULTS: At 9 weeks of age, both cortical and trabecular femoral bone volumes were increased in both male and female ObVDR-B6 mice, when compared to WT levels (P<0.05), without systemic changes to calciotropic parameters. The increase in femoral trabecular bone volume was associated with increase in MAR (P<0.01) and reduced osteoclast size (P<0.05). However, in female mice trabecular bone volume was unchanged in femoral metaphysis of 20 weeks mice and in vertebra both at 9 and 20 weeks of age. Increased cortical bone in both male and female ObVDR-B6 mice was due largely to increased periosteal expansion and was associated with increased cortical strength at 20 weeks of age. CONCLUSION: Overexpression of the human VDR gene in mature osteoblasts of C57Bl6/J mice increases cortical and trabecular bone volumes and confirms the previous reports of increased bone in OSVDR mice on the FVB/N background. However, site-specific and gender-related differences in bone volume suggest that the effects of osteoblast-specific VDR overexpression are more complex than hitherto recognised.

Vitamin D receptor overexpression in osteoblasts and osteocytes prevents bone loss during vitamin D-deficiency.

There are several lines of evidence that demonstrate the ability of 1,25-dihydroxyvitamin D (1,25(OH)2D3), acting via the vitamin D receptor (VDR) to mediate negative or positive effects in bone. Transgenic over-expression of VDR in osteoblasts and osteocytes in a mouse model (OSVDR) has been previously shown to inhibit processes of bone resorption and enhance bone formation, under conditions of adequate calcium intake. While these findings suggest that vitamin D signalling in osteoblasts and osteocytes promotes bone mineral accrual, the vitamin D requirement for this action is not well understood. In this study, 4 week old female OSVDR and wild-type (WT) mice were fed either a vitamin D-replete (1000IU/kg diet, D+) or vitamin D-deficient (D-) diet for 4 months to observe changes to bone mineral homeostasis. Tibial bone mineral volume was analysed by micro-CT and changes to bone cell activities were measured using standard dynamic histomorphometric techniques. While vitamin D-deplete WT mice demonstrated a reduction in periosteal bone accrual and overall bone mineral volume, OSVDR mice, however, displayed increased cortical and cancellous bone volume in mice which remained higher during vitamin D-depletion due to a reduced osteoclast number and increased bone formation rate. These data suggest that increased VDR-mediated activity in osteoblast and osteocytes prevents bone loss due to vitamin D-deficiency. This article is part of a Special Issue entitled '16th Vitamin D Workshop'.

Application of Holistic Liquid Chromatography-High Resolution Mass Spectrometry Based Urinary Metabolomics for Prostate Cancer Detection and Biomarker Discovery.

Human exhibit wide variations in their metabolic profiles because of differences in genetic factors, diet and lifestyle. Therefore in order to detect metabolic differences between individuals robust analytical methods are required. A protocol was produced based on the use of Liquid Chromatography- High Resolution Mass Spectrometry (LC-HRMS) in combination with orthogonal Hydrophilic Interaction (HILIC) and Reversed Phase (RP) liquid chromatography methods for the analysis of the urinary metabolome, which was then evaluated as a diagnostic tool for prostate cancer (a common but highly heterogeneous condition). The LC-HRMS method was found to be robust and exhibited excellent repeatability for retention times (<±1%), and mass accuracy (<±1 ppm). Based on normalised data (against creatinine levels, osmolality or MS total useful signals/MSTUS) coupled with supervised multivariate analysis using Orthogonal Partial Least Square-Discriminant Analysis (OPLS-DA), we were able to discriminate urine samples from men with or without prostate cancer with R2Y(cum) >0.9. In addition, using the receiver operator characteristics (ROC) test, the area under curve (AUC) for the combination of the four best characterised biomarker compounds was 0.896. The four biomarker compounds were also found to differ significantly (P<0.05) between an independent patient cohort and controls. This is the first time such a rigorous test has been applied to this type of model. If validated, the established protocol provides a robust approach with a potentially wide application to metabolite profiling of human biofluids in health and disease.

The pleiotropic effects of vitamin D in bone.

A current controversial question related to vitamin D supplementation is what level of serum 25-hydroxyvitamin D3 (25(OH)D3) is required to reduce the incidence of osteoporotic fractures. The reasoning behind vitamin D supplementation has been mostly derived from the role of vitamin D to promote intestinal calcium absorption and reduce bone resorption. While minimum 25(OH)D3 levels of 20nmol/L are required for sufficient intestinal calcium absorption to prevent osteomalacia, the mechanistic details of how higher 25(OH)D3 levels, well beyond that required for optimal calcium absorption, are able to prevent fractures and increase bone mineral density is unclear. Substantial evidence has arisen over the past decade that conversion of 25(OH)D3 to 1,25(OH)2D3via the 1-alpha hydroxylase (CYP27B1) enzyme in osteoblasts, osteocytes, chondrocytes and osteoclasts regulates processes such as cell proliferation, maturation and mineralization as well as bone resorption, which are all dependent on the presence the of the vitamin D receptor (VDR). We and others have also shown that increased vitamin D activity in mature osteoblasts by increasing levels of VDR or CYP27B1 leads to improved bone mineral volume using two separate transgenic mouse models. While questions remain regarding activities of vitamin D in bone to influence the anabolic and catabolic processes, the biological importance of vitamin D activity within the bone is unquestioned. However, a clearer understanding of the varied mechanisms by which vitamin D directly and indirectly influences mineral bone status are required to support evidence-based recommendations for vitamin D supplementation to reduce the risk of fractures. This article is part of a Special Issue entitled 'Vitamin D workshop'.

Robot-assisted radical prostatectomy in Hong Kong: a review of 235 cases.

OBJECTIVES: To report the outcome of all robot-assisted laparoscopic radical prostatectomy (RALP) in the public health care system in Hong Kong. PATIENTS AND METHODS: All patients who underwent RALP in the public health care system with at least 1 year of follow-up were evaluated. Data analysis included age, body mass index, preoperative prostate-specific antigen (PSA) level, D'Amico risk category, operative details, pathologic stage, follow-up continence, potency, and biochemical recurrence. RESULTS: Between 2005 and 2009, 235 patients underwent RALP, with a mean age of 66.4±5.9 years and a mean preoperative PSA level of 11.0±10.5 ng/mL. Complications were 16 (7%) in total. There were 176 (74.9%) patients with pT(2) disease and 55 (23.4%) patients with pT(3) disease. The overall rate of positive surgical margins (PSM) was 20.7%. At postoperative 12 months, 72.5% of the patients were pad free. For those 83 preoperative potent patients having nerve-sparing surgery, the overall trifecta rate at 12 months was 37.3%. Multivariate analysis identified that pathologic T staging was significantly associated with PSM, with an odds ratio (OR) of 7.884 (95% confidence interval [CI]: 3.576-17.379; P<0.001) for the pT(3) group compared with the pT(2) group. When comparing D'Amico medium- and high-risk categories with low-risk categories, they were found to be significantly associated with biochemical failure (medium- compared with low-risk: OR=3.536, 95% CI: 1.253-10.173, P=0.016; high- compared with low-risk: OR=10.214, 95% CI: 2.958-35.274, P<0.001). CONCLUSIONS: Our data demonstrate the feasibility, safety, and efficacy of RALP in low-to-intermediate volume centers. Our early oncologic outcomes were significantly correlated with pathologic stage and D'Amico risk stratification.

Clinical characteristics of childhood erythema multiforme, Stevens-Johnson syndrome and toxic epidermal necrolysis in Taiwanese children.

Erythema multiforme (EM), Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are mucocutaneous diseases associated with significant morbidity and mortality. This study compared childhood EM, SJS and TEN in terms of clinical courses, laboratory data, etiologies and outcomes in Taiwan. The initial laboratory findings, clinical presentations, etiologies and subsequent clinical courses of 30 patients with a diagnosis of EM, SJS or TEN, who were admitted between 1995 and 2003 at National Taiwan University Hospital were included and analyzed. There were 19 cases of EM, 8 cases of SJS, 2 cases of SJS/TEN and 1 case of TEN. The most common etiology in EM was infection (84.2%), and the most common implicated organism was Mycoplasma pneumoniae (42.1%). In contrast, 75% of SJS and 100% of TEN were induced by drugs. The most common offending drug was carbamazepine. Those patients with underlying diseases had more protracted courses and longer hospitalization stays. No mortalities were found in our cases. Early short-term steroid equivalent to 1-2 mg/kg/day of prednisolone for 3-5 days was used in 87.5% of SJS patients, without any significant side effects. Those with poor responsiveness to steroids and protracted courses were treated with additional intravenous immunoglobulin (IVIG) [1 g/kg/day], with satisfactory results. Early ophthalmic consultations were performed in all cases. No ocular complications were found in our cases. In conclusion, EM, SJS and TEN were associated with significant morbidity. Early ophthalmic consultations and withdrawal of the offending medication was necessary. Early short-term use of steroids in SJS showed promising results without significant side effects. The additional IVIG in those who had a poor response to steroid treatment may be helpful.