Evaluation of the potential of Delta-aminolevulinic acid for simultaneous detection of bioburden and anti-microbial photodynamic therapy of MRSA infected wounds in Swiss albino mice.

BACKGROUND: The dramatic increase of drug-resistant bacteria necessitates urgent development of platforms to simultaneously detect and inactivate bacteria causing wound infections, but are confronted with various challenges. Delta amino levulinic acid (ALA) induced protoporphyrin IX (PpIX) can be a promising modality for simultaneous bioburden diagnostics and therapeutics. Herein, we report utility of ALA induced protoporphyrin (PpIX) based simultaneous bioburden detection, photoinactivation and therapeutic outcome assessment in methicillin resistant Staphylococcus aureus (MRSA) infected wounds of mice. METHODS: MRSA infected wounds treated with 10% ALA were imaged with help of a blue LED (∼405 nm) based, USB powered, hand held device integrated with a modular graphic user interface (GUI). Effect of ALA application time, bacteria load, post bacteria application time points on wound fluorescence studied. PpIX fluorescence observed after excitation with blue LEDs was used to detect bioburden, start red light mediated antimicrobial photodynamic therapy (aPDT), determine aPDT effectiveness and assess selectivity of the approach. RESULTS: ALA-PpIX fluorescence of wound bed discriminates infected from uninfected wounds and detects clinically relevant load. While wound fluorescence pattern changes as a function of ALA incubation and post infection time, intra-wound inhomogeneity in fluorescence correlates with the Gram staining data on presence of biofilms foci. Lack of red fluorescence from wound granulation tissue treated with ALA suggests selectivity of the approach. Further, significant reduction (∼50%) in red fluorescence, quantified using the GUI, relates well with bacteria load reduction observed post topical aPDT. CONCLUSION: The potential of ALA induced PpIX for simultaneous detection of bioburden, photodynamic inactivation and "florescence-guided aPDT assessment" is demonstrated in MRSA infected wounds of mice.

National Control Laboratories in quality healthcare-a roadblock for substandard biopharmaceuticals.

Substandard or spurious drugs are a global problem with respect to Health and Economic burden. The impact is higher when medicines are from the category of life-saving drugs, essential medicines or high cost targeted medical treatment. Biopharmaceuticals are one such class of drugs where Quality testing plays a pivotal role to stop substandard drugs from reaching the patient. This study of 17,451 samples has highlighted the trend of occurrence of substandard biologicals (2.34%) over a decade (2011-2021) and the importance of continuous and complete evaluation of such Biopharmaceuticals. More such National Control Laboratories (NCL) should be involved in cross-checking the quality of the increasing number of biopharmaceuticals present in the market which are released only on the basis of the onsite inspection and dossier reviews. This will help the Regulators to ensure the readiness for testing the newer biologicals, devise effective policies for better health care initiatives and keep the substandard biopharmaceuticals at bay.

Nanotrap-Enhanced Raman Spectroscopy: An Efficient Technique for Trace Detection of Bioanalytes.

Reliable diagnosis of disease using body fluids requires sensitive and accurate detection of disease-specific analytes present in the fluid. In recent years, there has been increasing interest in using surface-enhanced Raman spectroscopy (SERS) for this purpose. The demonstrable signal enhancement and sensitivity of SERS makes it ideally suited for detection of a trace quantity of any analyte. However, lack of reproducibility along with large spatial variability in the measured Raman intensities due to differential (and often random) distribution of surface "hot spots" limits its routine clinical use. We propose here a technique, nanotrap-enhanced Raman spectroscopy (NTERS), for overcoming these long-standing limitations and challenges of SERS. In this technique, hot spots are formed by drying up a microvolume drop of the liquid, containing the mixture of nanoparticles and analytes in the focal volume of the Raman excitation laser, and the Raman signal is detected from these spots containing the analytes localized within the nanoparticle aggregates. The performance of the technique was evaluated in detecting trace quantities of two Raman-active analytes, Rhodamine 6G (R6G) and urea. It was found that R6G and urea could be detected down to a concentration of 50 nM with signal-to-noise ratio (SNR) value of ∼75 and 4 mM with SNR value of ∼500, respectively. A comparison with SERS revealed that NTERS not only had significantly superior (around 2 orders of magnitude) signal enhancement but also had high reproducibility because of its intrinsic ability to form nanoparticle aggregates with high repetitiveness. Another advantage of NTERS is its simplicity and cost effectiveness as it does not require any specialized substrate.

Effect of the polyelectrolyte coating on the photothermal efficiency of gold nanorods and the photothermal induced cancer cell damage.

Coating gold nanorods (GNRs) with polyelectrolytes is an effective approach to make them biocompatible for potential use in photothermal treatment (PTT) of cancer. The authors report the effect of coating of the GNRs with polystyrene sulphonate (PSS-GNRs) and PSS plus poly di-allyl di-methyl ammonium chloride (PDDAC-GNRs) on its photothermal conversion efficiency (PTE), cellular uptake and subsequently the photothermal induced cytotoxicity in human oral cancer cells (NT8e). Coating of GNRs with PSS led to decrease in PTE by ∼30% and further coating it with PDDAC led to its increase to similar level, with respect to as- prepared GNRs. The cellular uptake of PDDAC-GNRs in cancer cells was double than that for PSS-GNRs. PTT of cancer cells after treatment with 60 pM of either PDDAC-GNRs or PSS-GNRs resulted in cytotoxicty of ∼90%. At higher concentration of 120 pM, while PSS-GNRs showed no further change, for PDDAC-GNR the photothermal induced cytotoxicity decreased to ∼50%. The broadening of longitudinal surface plasmon peak of PDDAC-GNRs and appearance of dark clusters in cells under bright-field microscope suggested intracellular clustering of PDDAC-GNRs. In conclusion, despite high PTE and cellular uptake of PDDAC-GNRs, its intracellular clustering (due to acidic pH ) adversely affect the PTT of cancer cells.

Intra- and intermolecular domain interactions among novel two-component system proteins coded by Rv0600c, Rv0601c and Rv0602c of Mycobacterium tuberculosis.

Two-component signal transduction pathways comprising a histidine kinase and its cognate response regulator play a dominant role in the adaptation of Mycobacterium tuberculosis to its host, and its virulence, pathogenicity and latency. Autophosphorylation occurs at a conserved histidine of the histidine kinase and subsequently the phosphoryl group is transferred to the conserved aspartate of its cognate response regulator. Among the twelve two-component systems of M. tuberculosis, Rv0600c (HK1), Rv0601c (HK2) and Rv0602c (TcrA) are annotated as a unique three-protein two-component system. HK1 contains an ATP-binding domain, and HK2, a novel Hpt mono-domain protein, contains the conserved phosphorylable histidine residue. HK1 and HK2 complement each other's functions. Interactions among different domains of the HK1, HK2 and TcrA proteins were studied using a yeast two-hybrid system. Self-interaction was observed for HK2 but not for HK1 or TcrA. HK2 was found to interact reasonably well with both HK1 and TcrA, but HK1 interacted weakly with TcrA. The conserved aspartate-containing receiver domain of TcrA interacted well with HK2 but not with HK1. These results suggest the existence of a novel signalling mechanism amongst HK1-HK2-TcrA, and a model for this mechanism is proposed.

Probing the nucleotide binding and phosphorylation by the histidine kinase of a novel three-protein two-component system from Mycobacterium tuberculosis.

The two-component signal transduction system from Mycobacterium tuberculosis bears a unique three-protein system comprising of two putative histidine kinases (HK1 and HK2) and one response regulator TcrA. By sequence analysis, HK1 is found to be an adenosine 5'-triphosphate (ATP) binding protein, similar to the nucleotide-binding domain of homologous histidine kinases, and HK2 is a unique histidine containing phosphotransfer (HPt)-mono-domain protein. HK1 is expected to interact with and phosphorylate HK2. Here, we show that HK1 binds 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate monolithium trisodium salt and ATP with a 1:1 stoichiometric ratio. The ATPase activity of HK1 in the presence of HK2 was measured, and phosphorylation experiments suggested that HK1 acts as a functional kinase and phosphorylates HK2 by interacting with it. Further phosphorylation studies showed transfer of a phosphoryl group from HK2 to the response regulator TcrA. These results indicate a new mode of interaction for phosphotransfer between the two-component system proteins in bacteria.

Temperature and urea induced conformational changes of the histidine kinases from Mycobacterium tuberculosis.

A unique three protein two-component system is present in Mycobacterium tuberculosis comprising of two histidine kinases (Rv0600c/HK1 and Rv0601c/HK2) and a response regulator (Rv0602c/TcrA). The HK2 is a novel HPt-mono domain protein absent in other bacteria. We present here the temperature and urea induced denaturation study of HK1 and HK2 using circular dichroism and fluorescence spectroscopy. HK1 and HK2 are thermally quite stable. Thermal transition of HK1 is a two-state process and that of HK2 is a three-state process. Urea denaturation of HK1 and HK2 is a three-state and two-state process, respectively. The DeltaG degrees of the two transitions during urea induced unfolding of HK1 is 4.76+/-0.6 kcal/mol and -7.11+/-0.8 kcal/mol. Unfolding of HK2 in presence of urea has DeltaG degrees of 4.766+/-0.5 kcal/mol. The intrinsic fluorescence study of HK2 unfolding implies flexibility of proline rich loop in the tryptophan bearing HAMP domain.

Functional insights from the molecular modelling of a novel two-component system.

Two-component systems (TCSs) are the major signalling pathway in bacteria and represent potential drug targets. Among the 11 paired TCS proteins present in Mycobacterium tuberculosis H37Rv, the histidine kinases (HKs) Rv0600c (HK1) and Rv0601c (HK2) are annotated to phosphorylate one response regulator (RR) Rv0602c (TcrA). We wanted to establish the sequence-structure-function relationship to elucidate the mechanism of phosphotransfer using in silico methods. Sequence alignments and codon usage analysis showed that the two domains encoded by a single gene in homologous HKs have been separated into individual open-reading frames in M. tuberculosis. This is the first example where two incomplete HKs are involved in phosphorylating a single RR. The model shows that HK2 is a unique histidine phosphotransfer (HPt)-mono-domain protein, not found as lone protein in other bacteria. The secondary structure of HKs was confirmed using "far-UV" circular dichroism study of purified proteins. We propose that HK1 phosphorylates HK2 at the conserved H131 and the phosphoryl group is then transferred to D73 of TcrA.