Plasmodium falciparum AMA1 and CSP antigen diversity in parasite isolates from southern Ghana.

Introduction: Diversity in malarial antigens is an immune evasion mechanism that gives malaria parasites an edge over the host. Immune responses against one variant of a polymorphic antigen are usually not fully effective against other variants due to altered epitopes. This study aimed to evaluate diversity in the Plasmodium falciparum antigens apical membrane antigen 1 (PfAMA1) and circumsporozoite protein (PfCSP) from circulating parasites in a malaria-endemic community in southern Ghana and to determine the effects of polymorphisms on antibody response specificity. Methods: The study involved 300 subjects, whose P. falciparum infection status was determined by microscopy and PCR. Diversity within the two antigens was evaluated by msp2 gene typing and molecular gene sequencing, while the host plasma levels of antibodies against PfAMA1, PfCSP, and two synthetic 24mer peptides from the conserved central repeat region of PfCSP, were measured by ELISA. Results: Of the 300 subjects, 171 (57%) had P. falciparum infection, with 165 of the 171 (96.5%) being positive for either or both of the msp2 allelic families. Gene sequencing of DNA from 55 clonally infected samples identified a total of 56 non-synonymous single nucleotide polymorphisms (SNPs) for the Pfama1 gene and these resulted in 44 polymorphic positions, including two novel positions (363 and 365). Sequencing of the Pfcsp gene from 69 clonal DNA samples identified 50 non-synonymous SNPs that resulted in 42 polymorphic positions, with half (21) of these polymorphic positions being novel. Of the measured antibodies, only anti-PfCSP antibodies varied considerably between PCR parasite-positive and parasite-negative persons. Discussion: These data confirm the presence of a considerable amount of unique, previously unreported amino acid changes, especially within PfCSP. Drivers for this diversity in the Pfcsp gene do not immediately seem apparent, as immune pressure will be expected to drive a similar level of diversity in the Pfama1 gene.

Production and Purification of Plasmodium Circumsporozoite Protein in Lactococcus lactis.

Malaria is a vector-borne disease caused by Plasmodium parasites of which Plasmodium falciparum contributed to an estimated 247 million cases worldwide in 2021 (WHO malaria report 2022). The P. falciparum Circumsporozoite protein (PfCSP) covers the surface of the sporozoite which is critical to cell invasion in the human host. PfCSP is the leading pre-erythrocytic vaccine candidate and forms the basis of the RTS'S (Mosquirix®) malaria vaccine. However, high-yield production of full-length PfCSP with proper folding has been challenging. Here, we describe expression and purification of full-length PfCSP (containing 4 NVDP and 38 NANP repeats) with proper conformation by a simple three-step procedure in the Lactococcus lactis expression system.

Clinical formulation development of Plasmodium falciparum malaria vaccine candidates based on Pfs48/45, Pfs230, and PfCSP.

Two malaria transmission-blocking vaccine (TBV) candidates, R0.6C and ProC6C, have completed preclinical development including the selection of adjuvants, Alhydrogel® with or without the saponin based adjuvant Matrix-M™. Here, we report on the final drug product (formulation) design of R0.6C and ProC6C and evaluate their safety and biochemical stability in preparation for preclinical and clinical pharmacy handling. The point-of-injection stability studies demonstrated that both the R0.6C and ProC6C antigens are stable on Alhydrogel in the presence or absence of Matrix-M for up to 24 h at room temperature. As this is the first study to combine Alhydrogel and Matrix-M for clinical use, we also evaluated their potential interactions. Matrix-M adsorbs to Alhydrogel, while not displacing the > 95 % adsorbed protein. The R0.6C and ProC6C formulations were found to be safe and well tolerated in repeated dose toxicity studies in rabbits generating high levels of functional antibodies that blocked infection of mosquitoes. Further, the R0.6C and ProC6C drug products were found to be stable for minimally 24 months when stored at 2-8 °C, with studies ongoing through 36 months. Together, this data demonstrates the safety and suitability of the L. lactis expression system as well as supports the clinical testing of the R0.6C and ProC6C malaria vaccine candidates in First-In-Human clinical trials.

A randomized first-in-human phase I trial of differentially adjuvanted Pfs48/45 malaria vaccines in Burkinabé adults.

BACKGROUNDMalaria transmission-blocking vaccines aim to interrupt the transmission of malaria from one person to another.METHODSThe candidates R0.6C and ProC6C share the 6C domain of the Plasmodium falciparum sexual-stage antigen Pfs48/45. R0.6C utilizes the glutamate-rich protein (GLURP) as a carrier, and ProC6C includes a second domain (Pfs230-Pro) and a short 36-amino acid circumsporozoite protein (CSP) sequence. Healthy adults (n = 125) from a malaria-endemic area of Burkina Faso were immunized with 3 intramuscular injections, 4 weeks apart, of 30 µg or 100 µg R0.6C or ProC6C each adsorbed to Alhydrogel (AlOH) adjuvant alone or in combination with Matrix-M (15 µg or 50 µg, respectively). The allocation was random and double-blind for this phase I trial.RESULTSThe vaccines were safe and well tolerated with no vaccine-related serious adverse events. A total of 7 adverse events, mild to moderate in intensity and considered possibly related to the study vaccines, were recorded. Vaccine-specific antibodies were highest in volunteers immunized with 100 µg ProC6C-AlOH with Matrix-M, and 13 of 20 (65%) individuals in the group showed greater than 80% transmission-reducing activity (TRA) when evaluated in the standard membrane feeding assay at 15 mg/mL IgG. In contrast, R0.6C induced sporadic TRA.CONCLUSIONAll formulations were safe and well tolerated in a malaria-endemic area of Africa in healthy adults. The ProC6C-AlOH/Matrix-M vaccine elicited the highest levels of functional antibodies, meriting further investigation.TRIAL REGISTRATIONPan-African Clinical Trials Registry (https://pactr.samrc.ac.za) PACTR202201848463189.FUNDINGThe study was funded by the European and Developing Countries Clinical Trials Partnership (grant RIA2018SV-2311).

Nontargeted Metabolite Profiling of the Most Prominent Indian Mango (Mangifera indica L.) Cultivars Using Different Extraction Methods.

Aroma has a crucial role in assessing the quality of fresh fruit and its processed versions, which serve as reliable indications for advancing local cultivars in the mango industry. The aroma of mango is attributed to a complex of hundreds of volatile, polar, and nonpolar metabolites belonging to different chemical classes like monoterpenes, sesquiterpenes, nonterpene hydrocarbons (alkanes), alcohols, esters, fatty acids, aldehydes, lactones, amides, amines, ethers, and many more. This study looked at the volatile, nonpolar, and polar metabolites from 16 mango cultivars to determine their relative quantities and intervarietal changes using hexane, ethanol, and solid-phase microextraction (SPME), followed by gas chromatography-mass spectrometry (GC-MS) analysis. In total, 58 volatile compounds through SPME, 50 nonpolar metabolites from hexane extract, and 52 polar metabolites from ethanol extract were detected from all of the cultivars, belonging to various chemical classes. Through the SPME method, all 16 mango cultivars except Dashehari and Neelum exhibited abundant monoterpenes with maximum concentration in Kesar (91.00%) and minimum in Amrapali (60.66%). However, the abundance of fatty acids and sesquiterpenes was detected in Dashehari (37.91%) and Neelum (74.80%), respectively. In the hexane extract, 23 nonterpene hydrocarbons exhibited abundance in all 16 mango cultivars except Baneshan, with a higher concentration in Dashehari (95.45%) and lower in Ratna (77.63%). The ethanol extraction of 16 mango cultivars showed a higher concentration of esters, aldehydes, alcohols, and amides in Jamadar (52.16%), Dadamio (74.30%), Langra (64.38%), and Kesar (37.10%), respectively. There have been a lot of metabolite variations observed and analyzed using hierarchical cluster analysis (HCA) and principal component analysis (PCA) based on the similarity of various chemical compounds. Cluster analysis revealed the true similarity and pedigree of different mango cultivars, viz., Neeleswari, Dashehari, Neelum, Alphonso, Baneshan, Sonpari, and Neeleshan. They occupied the same cluster during analysis.

Serological evaluation of risk factors for exposure to malaria in a pre-elimination setting in Malaysian Borneo.

Malaysia has reported no indigenous cases of P. falciparum and P. vivax for over 3 years. When transmission reaches such low levels, it is important to understand the individuals and locations where exposure risks are high, as they may be at greater risk in the case of a resurgence of transmission. Serology is a useful tool in low transmission settings, providing insight into exposure over longer durations than PCR or RDT. We ran blood samples from a 2015 population-based survey in northern Sabah, Malaysian Borneo on a multiplex bead assay. Using supervised machine learning methods, we characterised recent and historic exposure to Plasmodium falciparum and P. vivax and found recent exposure to P. falciparum to be very low, with exposure to both species increasing with age. We performed a risk-factor assessment on environmental, behavioural, demographic and household factors, and identified forest activity and longer travel times to healthcare as common risk-factors for exposure to P. falciparum and P. vivax. In addition, we used remote-sensing derived data and geostatistical models to assess environmental and spatial associations with exposure. We created predictive maps of exposure to recent P. falciparum in the study area and showed 3 clear foci of exposure. This study provides useful insight into the environmental, spatial and demographic risk factors for P. falciparum and P. vivax at a period of low transmission in Malaysian Borneo. The findings would be valuable in the case of resurgence of human malarias in the region.

High opsonic phagocytosis activity and growth inhibition of merozoites are associated with RON4 antibody levels and protect against febrile malaria in Ghanaian children.

Background: Naturally acquired immunity to malaria may involve different immune mechanisms working in concert, however, their respective contributions and potential antigenic targets have not been clearly established. Here, we assessed the roles of opsonic phagocytosis and antibody-mediated merozoite growth inhibition in Plasmodium falciparum (P. falciparum) infection outcomes in Ghanaian children. Methods: The levels of merozoite opsonic phagocytosis, growth inhibition activities and six P. falciparum antigen-specific IgG of plasma samples from children (n=238, aged 0.5 to 13 years) were measured at baseline prior to the malaria seasons in southern Ghana. The children were then actively and passively followed up for febrile malaria and asymptomatic P. falciparum infection detection in a 50-week longitudinal cohort. P. falciparum infection outcome was modelled as a function of the measured immune parameters while accounting for important demographic factors. Results: High plasma activity of opsonic phagocytosis [adjusted odds ratio (aOR)= 0.16; 95%CI= 0.05 - 0.50, p = 0.002], and growth inhibition (aOR=0.15; 95% CI = 0.04-0.47; p = 0.001) were individually associated with protection against febrile malaria. There was no evidence of correlation (b= 0.13; 95% CI= -0.04-0.30; p=0.14) between the two assays. IgG antibodies against MSPDBL1 correlated with opsonic phagocytosis (OP) while IgG against PfRh2a correlated with growth inhibition. Notably, IgG antibodies against RON4 correlated with both assays. Conclusion: Opsonic phagocytosis and growth inhibition are protective immune mechanisms against malaria that may be acting independently to confer overall protection. Vaccines incorporating RON4 may benefit from both immune mechanisms.

The use of augmentative and alternative communication by children with developmental disability in the classroom: a case study.

PURPOSE: Augmentative and alternative communication (AAC) systems are often introduced to children with disabilities who demonstrate complex communication needs. As attending school is an essential part of these children's lives, it is important that they use their AAC system to communicate in the classroom. This study aimed to describe the nature of the use of AAC by students with developmental disabilities in the classroom. MATERIALS AND METHOD: This study was conducted in Malaysia. Six students were observed twice each in their classroom and their classroom interactions were video recorded. The video recordings were transcribed and coded for the presence of a communication event, the student's mode of communication and communication function, the communication partner involved, and access to the AAC system. RESULTS: Contrary to past studies, most students in this study spontaneously initiated interaction almost as many times as they responded. They primarily communicated with gestures and verbalizations/vocalizations despite having been introduced to an AAC system. When students communicated using their AAC system, they mainly interacted with the teachers, and for the function of either behavioral regulation or joint attention. It was found that for 39% of communicative events, the student's aided AAC system was not within arm's reach. CONCLUSION: The findings highlight the need for efforts to encourage students with complex communication needs to use AAC more frequently in their classroom to be able to communicate more effectively and for a wider range of communicative functions. Speech-language pathologists can work closely with teachers to provide the necessary support to these students.

Speech-language pathologists (SLPs) can provide ongoing support to school teachers to equip them with the necessary knowledge and skills to support the use of augmentative and alternative communication (AAC) in the classroom.SLPs can also focus on teaching students to use AAC for a variety of communication functions and with various partners rather than only adult communication partners.

Dissipation kinetics and health risk assessment of certain insecticides applied in/on tomato under open field and poly-house conditions.

In this study, the dissipation kinetics and health risk assessment of different insecticides in tomato under open field and poly-house conditions were investigated. A total of four insecticides, namely Chlorantraniprole 18.5 SC @ 30 g a.i ha-1, Flubendiamide 20% WG @ 48.0 g a.i ha-1, Indoxacarb 14.5 SC @ 60.0 g a.i ha-1, and Thiamethoxam 25% WG @ 50.0 g a.i ha-1, were applied on tomato at the 50% flowering stage, followed by 10 days after the first spray. Prior to actual sample analysis, QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) based extraction methodology for the chlorantraniliprole, flubendiamide, indoxacarb and thiamethoxam in tomato were verified and quantified on ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) in terms of linearity, sensitivity (detection limits), accuracy (% recovery) and precision (% RSD). The DT50 value of chlorantraniliprole, flubendiamide, indoxacarb and thiamethoxam in tomato under open field condition was 1.95, 2.25, 2.37 and 3.98, respectively and under poly house condition it was 3.05, 5.02, 2.68 and 4.82, respectively. Similarly, the safe waiting period of chlorantraniliprole, flubendiamide, indoxacarb and thiamethoxam in tomato under open field condition was 1.05, 0.83, 2.96 and 3.23, respectively and under poly house condition it was 2.02, 4.93, 4.09 and 7.33, respectively. Further, health risk assessment was evaluated and observed no risk for Indian consumers due to application of studied insecticides (RQ < 1) under open field and poly-house conditions.

Cloning and Recombinant Protein Expression in Lactococcus lactis.

The Lactococcus lactis, a Gram-positive bacteria, is an ideal expression host for the overproduction of heterologous proteins in a properly folded and functional form. L. lactis has been identified as an efficient cell factory, generally recognized as safe (GRAS), has a long history of safe use in food production, and is known to have probiotic properties. Key desirable features of L. lactis include the following: (1) rapid growth to high cell densities, not requiring aeration which facilitates large-scale fermentation; (2) its Gram-positive nature precludes the presence of contaminating endotoxins; (3) the capacity to secrete stable recombinant protein into the growth medium with few proteases resulting in a properly folded, full-length protein; and (4) the availability of diverse expression vectors facilitating various cloning options. We have previously described production of several recombinant proteins with varying degrees of predicted structural complexities using the L. lactis pH-dependent P170 promoter. The purpose of this chapter is to provide a detailed protocol for facilitating wider application of L. lactis as a reliable platform for expression of heterologous recombinant proteins in soluble form. Here, we present details of the various steps involved such as cloning of the target gene in appropriate expression plasmid vector, determination of the expression levels of the heterologous protein, and initial purification of the expressed soluble recombinant protein of interest.

B-Cell Epitope Mapping of the Plasmodium falciparum Malaria Vaccine Candidate GMZ2.6c in a Naturally Exposed Population of the Brazilian Amazon.

The GMZ2.6c malaria vaccine candidate is a multi-stage P. falciparum chimeric protein that contains a fragment of the sexual-stage Pfs48/45-6C protein genetically fused to GMZ2, an asexual-stage vaccine construction consisting of the N-terminal region of the glutamate-rich protein (GLURP) and the C-terminal region of the merozoite surface protein-3 (MSP-3). Previous studies showed that GMZ2.6c is widely recognized by antibodies from Brazilian exposed individuals and that its components are immunogenic in natural infection by P. falciparum. In addition, anti-GMZ2.6c antibodies increase with exposure to infection and may contribute to parasite immunity. Therefore, identifying epitopes of proteins recognized by antibodies may be an important tool for understanding protective immunity. Herein, we identify and validate the B-cell epitopes of GMZ2.6c as immunogenic and immunodominant in individuals exposed to malaria living in endemic areas of the Brazilian Amazon. Specific IgG antibodies and subclasses against MSP-3, GLURP, and Pfs48/45 epitopes were detected by ELISA using synthetic peptides corresponding to B-cell epitopes previously described for MSP-3 and GLURP or identified by BepiPred for Pfs48/45. The results showed that the immunodominant epitopes were P11 from GLURP and MSP-3c and DG210 from MSP-3. The IgG1 and IgG3 subclasses were preferentially induced against these epitopes, supporting previous studies that these proteins are targets for cytophilic antibodies, important for the acquisition of protective immunity. Most individuals presented detectable IgG antibodies against Pfs48/45a and/or Pfs48/45b, validating the prediction of linear B-cell epitopes. The higher frequency and antibody levels against different epitopes from GLURP, MSP-3, and Pfs48/45 provide additional information that may suggest the relevance of GMZ2.6c as a multi-stage malaria vaccine candidate.

An immunoinformatic approach towards development of a potent and effective multi-epitope vaccine against monkeypox virus (MPXV).

Monkeypox is a viral zoonotic disease, often transmitted to humans from animals. While the whole world is haggling with the COVID-19 pandemic, the emergence of the monkeypox virus (MPXV) arose as a new challenge to mankind. Till date, numerous cases related to the MPXV have been reported in several countries across the globe, but, its momentary distribution in the current time has left everyone in fright with increasing mortality and limited clinically approved treatments. Therefore, it is of immense importance to develop a potent and highly effective vaccine capable of inducing desired immunogenic responses against the highly contagious MPXV. Herein, using various immunoinformatic and computational biology tools, we made an attempt to develop a multi-epitope vaccine construct against the MPXV which is antigenic, non-allergen and non-toxic in nature and capable of exhibiting immunogenic behavior. The sequence of vaccine construct was designed using the proposed 4 MHC-I, 3 MHC-II and 4 B-cell epitopes linked with suitable adjuvant and linkers. The modeled structure of the vaccine construct was used to assess its interaction with the Toll-like Receptor 4 (TLR4) using ClusPro and HADDOCK. All-atoms molecular dynamics simulation of the MPXV vaccine construct-TLR4 complex followed by a high level of gene expression of the construct within the bacterial system affirmed its stability along with induction of immunogenic response within the host cell. Altogether, our immunoinformatic approach aid in the development of a stable chimeric vaccine construct against MPXV and needs further experimental validation for its immunological relevance and usefulness as a vaccine candidate.Communicated by Ramaswamy H. Sarma.

Parents' perception on the use of augmentative and alternative communication by children with complex communication needs in Malaysia.

PURPOSE: Children with developmental disabilities who have complex communication needs (CCN) frequently need to use augmentative and alternative communication (AAC) to communicate effectively and efficiently. Speech-language pathologists (SLPs) often work closely with parents and other professionals when deciding on the best AAC system to introduce to these children. This study aimed to describe the use of AAC by children with CCN in Malaysia as reported by their parents. MATERIALS AND METHOD: An online survey distributed for this study was completed by 235 parents. RESULTS: Most of the parents of children with CCN who participated in this study reported that their children used low-tech AAC systems. A majority of respondents were satisfied with their child's AAC system. Parental satisfaction was positively associated with the frequency of use and whether the use of AAC helped parents understand the child better. Challenges reported by parents when using AAC and the reason some families abandoned the use of AAC were similar. Examples of challenges include parents having limited time and the child lacking the motivation to use the AAC system. CONCLUSION: The findings of this study suggest the importance of SLPs actively involving parents in the selection of their children's AAC system so they are agreeable with the system introduced and continuously supporting children and their families to encourage and sustain the use of AAC. Implications for rehabilitationSpeech-language pathologists (SLPs) can create communication opportunities for the child to use augmentative and alternative communication (AAC) and experience success, teach parents how to incorporate AAC into the family's daily routine and activities, and reduce the demands on parents by preparing the AAC materials and programming the AAC system where possible.SLPs can provide ongoing support to school teachers to equip them with the necessary knowledge and skills to support the use of AAC in the classroom.

The relationship between affiliate stigma, stress, and quality of life for parents of individuals with cerebral palsy in Malaysia.

OBJECTIVE: To examine the relationship between affiliate stigma, stress, and perceived quality of life among parents of children with cerebral palsy (CP). METHOD: Surveys (Study 1) and semi-structured interviews (Study 2) were used to collect data. Thirty-eight respondents completed the Affiliate Stigma Scale (ASS), Caregiver Burden Inventory (CBI), and Care-related Quality of Life (CarerQol) scales (GMFCS). Eleven respondents took part in semi-structured interviews, which were then thematically evaluated. RESULTS: Parents did not feel stigmatized because they had a child with CP. However, parents needed some form of short-term relief from caregiving. After controlling for demographics, high stress respondents experienced high affiliate stigma whereas low stress respondents had better quality of life. Although the respondents with less stigma had a better quality of life, this effect was moderate. Three major themes emerged from the interviews. CONCLUSION: Counseling, support groups, and helper services should be offered to parents. Also, healthcare professionals participating in CP rehabilitation, community-based rehabilitation, and health institutions should be educated on how to better help parents of children with CP.

There is a need in Malaysia to offer counseling, support groups, and assistance programs for parents of children with cerebral palsy.Healthcare professionals, who offer rehabilitation services to children with cerebral palsy, need greater awareness of the needs of families and caregivers and should be educated about how to best support them.There should be greater recognition of the importance of respite and relief from caregiving among Malaysian parents of children with cerebral palsy.

Development of a novel method for multiple phytohormone analysis by UHPLC-MS/MS from bio-enriched organic fertilizer prepared using banana pseudostem sap waste.

Banana harvesting generates a large amount of banana pseudostem waste, which is generally burnt or thrown away, despite containing many nutrients. Bio-enriched organic fertilizer (BOF) was prepared from banana pseudostem sap (BPS), and it has been patented (Patent No. WO 2013/001478 Al). Several reports revealed that its application increases plant growth promotion of various horticulture crops. Apart from macro- and micronutrients, it also contained phytohormones. Hence, the present study aims to detect and quantify phytohormone in it. A novel method was developed to extract four phytohormones, viz., indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), gibberellic acid (GA3), and salicylic acid (SA) using single solvent from BPS and BOF. Extracted hormones were analyzed by ultrahigh-performance liquid chromatography coupled with heated electrospray ionization tandem mass spectrometry (UHPLC-HESI-MS/MS). BOF showed a higher concentration of IAA, IBA, GA3, and SA than BPS. Thus, this is the first time a method has been reported to extract and detect phytohormones from banana pseudostem sap.

Evaluation of Ozonation Technique for Pesticide Residue Removal in Okra and Green Chili Using GC-ECD and LC-MS/MS.

The indiscriminate use of pesticides in agricultural commodities has become a global health concern. Various household methods are employed to remove pesticide residues from agricultural commodities, e.g., water and ozone. Many ozone-based commercial pesticide removal machines are available in the market for the general public. The current study compares the pesticide removal efficiency of ozone-based washing of fruits and vegetables to simple tap water through commercially available machines and its health risk assessment to different age groups of consumers. The okra and green chili fruits were treated with acetamiprid and ethion as foliar application at the fruiting stage, using the recommended dose (RD) and double to the recommended dose (2RD), respectively. A modified QuEChERS-based pesticide extraction method was verified for its accuracy, precision, linearity, and sensitivity. The treated samples were washed with tap and ozonated water at different intervals, i.e., 3, 8, and 10 min using a commercial food purifier. Washing with ozonized water for 3 min recorded the maximum removal of acetamiprid and ethion from okra and chili fruits. Further, the risk quotient values (RQ) obtained were lower than one at both doses. Thus, washing vegetables with ozonized water for 3 min ensures vegetables are safer for general consumption without any health risk to Indian consumers.

Malaysian speech-language pathology students' reflections about their participation in an AAC training program.

Most speech-language pathologists (SLPs) in Malaysia practice with an undergraduate degree, which provides them with limited knowledge about and training in augmentative and alternative communication (AAC). This limited knowledge and training may affect their confidence and competence when introducing and using AAC with individuals for whom it is required. This study aimed to obtain feedback, via semi-structured interviews, from a group of 11 Malaysian university students who participated in an AAC training program about their experiences participating in and the effectiveness of the training program. Three themes were derived from qualitative content analysis of the interviews: (a)Time Demands, (b) Generalizing the use of AAC, and (c) Learning Required When Introducing AAC. The participants reported that they obtained better insights into the role of SLPs and communication partners with regards to AAC, as well as the struggles they faced. Students also reported increased confidence when working with individuals who use AAC after participating in the training program, thus supporting the need for similar training programs in the future.

Pseudomonas aeruginosa inhibits quorum-sensing mechanisms of soft rot pathogen Lelliottia amnigena RCE to regulate its virulence factors and biofilm formation.

The quorum-sensing (QS) cascade is responsible for the colonization and phenotypic behavior of the pathogenic organism and the regulation of diverse signal molecules. The disruption of the quorum-sensing system is an effective strategy to overcome the possibility of antibiotic resistance development in the pathogen. The quorum quenching does not kill the microbes. Instead, it hinders the expression of pathogenic traits. In the present experiment, Pseudomonas aeruginosa RKC1 was used to extract the metabolites responsible for quorum-sensing inhibition in soft rot pathogen Lelliottia amnigena RCE. During the initial screening, P. aeruginosa RKC1 was found to be most promising and inhibits violacein of Chromobacterium violaceum MTCC2656 pyocyanin, swarming-swimming motility of P. aeruginosa MTCC2297. The characterization of metabolites produced by the microbes which are responsible for quorum-sensing inhibition through GC-MS is very scarce in scientific literature. The ethyl acetate extract of P. aeruginosa RKC1 inhibits biofilm formation of L. amnigena RCE while inhibiting growth at higher concentrations. The GC-MS analysis suggested that Cyclic dipeptides (CDPs) such as Cyclo (L-prolyl-L-valine), Cyclo (Pro-Leu), and Cyclo(D-phenylalanyl-L-prolyl) were predominantly found in the ethyl acetate extract of the P. aeruginosa RKC1 (93.72%). This diketopiperazine (DKPs) exhibited quorum-sensing inhibition against the pathogen in liquid media during the active growth phase and regulated diverse metabolites of the pathogen. Moreover, the metabolites data from the clear zone around wells showed a higher concentration of DKSs (9.66%) compared to other metabolites. So far, very few reports indicate the role of DKPs or CDPs in inhibiting the quorum-sensing system in plant pathogenic bacteria. This is one such report that exploits metabolites of P. aeruginosa RKC1. The present investigation provided evidence to use quorum-sensing inhibitor metabolites, to suppress microbes' pathogenesis and thus develop an innovative strategy to overcome antibiotic resistance.

Quorum Sensing Inhibitory and Quenching Activity of Bacillus cereus RC1 Extracts on Soft Rot-Causing Bacteria Lelliottia amnigena.

The quorum sensing (QS) system of bacteria helps them to communicate with each other in a density-dependent manner and regulates pathogenicity. The concentrations of autoinducers, peptides, and signaling factors are required for determining the expression of virulence factors in many pathogens. The QS signals of the pathogen are regulated by the signal transduction pathway. The binding of signal molecules to its cognate receptor brings changes in the structure of the receptor, makes it more accessible to the DNA, and thus regulates diverse expression patterns, including virulence factors. Degrading the autoinducer molecules or disturbing the quorum sensing network could be exploited to control the virulence of the pathogen while avoiding multidrug-resistant phenotypes. The rhizosphere is a tremendous source of beneficial microbes that has not yet been explored properly for its anti-quorum sensing potential. Lelliottia amnigena causes soft rot diseases in onion, potato, and other species. The present investigation was carried out with the aim of isolating the anti-quorum sensing metabolites and elucidating their role in controlling the virulence factors of the pathogen by performing a maceration assay. The ethyl acetate extracts of various bacteria are promising for violacein inhibition assay using Chromobacterium violaceum MTCC2656 and pyocyanin inhibition of Pseudomonas aeruginosa MTCC2297. Therefore, the extract was used to deduce its role in attenuation of soft rot in potato, carrot, and cucumber. The maximum reduction of macerated tissue in carrot, potato, and cucumber was given by Bacillus cereus RC1 at 91.22, 97.59, and 88.78%, respectively. The concentration-dependent inhibition of virulence traits was observed during the entire experiment. The quorum quenching potential of the bacterial extract was used to understand the regulatory metabolites. The data of the diffusible zone and gas chromatography-mass spectrometry (GC-MS) analysis showed that diketopiperazines, viz. Cyclo(d-phenylalanyl-l-prolyl), Cyclo Phe-Val, Cyclo(Pro-Ala), Cyclo(l-prolyl-l-valine), Cyclo (Leu-Leu), and Cyclo(-Leu-Pro), are prominent metabolites that could modulate the pathogenicity in L. amnigena RCE. The interaction of bacterial extracts regulates various metabolites of the pathogens during their growth in liquid culture compared to their control counterparts. This study might help in exploiting the metabolites from bacteria to control the pathogens, with concurrent reduction in the pathogenicity of the pathogens without developing antibiotic resistance.

Heterologous Expression and Evaluation of Novel Plasmodium falciparum Transmission Blocking Vaccine Candidates.

Malaria transmission blocking vaccines (TBV) aim to induce antibodies that can interrupt Plasmodium falciparum development in the mosquito midgut and thereby prevent onward malaria transmission. A limited number of TBV candidates have been identified and only three (Pfs25, Pfs230 and Pfs48/45) have entered clinical testing. While one of these candidates may emerge as a highly potent TBV candidate, it is premature to determine if they will generate sufficiently potent and sustained responses. It is therefore important to explore novel candidate antigens. We recently analyzed sera from naturally exposed individuals and found that the presence and/or intensity of antibodies against 12 novel putative surface expressed gametocyte antigens was associated with transmission reducing activity. In this study, protein fragments of these novel TBV candidates were designed and heterologously expressed in Drosophila melanogaster S2 cells and Lactococcus lactis. Eleven protein fragments, covering seven TBV candidates, were successfully produced. All tested antigens were recognized by antibodies from individuals living in malaria-endemic areas, indicating that native epitopes are present. All antigens induced antigen-specific antibody responses in mice. Two antigens induced antibodies that recognized a native protein in gametocyte extract, and antibodies elicited by four antigens recognized whole gametocytes. In particular, we found that antigen Pf3D7_0305300, a putative transporter, is abundantly expressed on the surface of gametocytes. However, none of the seven novel TBV candidates expressed here induced an antibody response that reduced parasite development in the mosquito midgut as assessed in the standard membrane feeding assay. Altogether, the antigen fragments used in this study did not prove to be promising transmission blocking vaccine constructs, but led to the identification of two gametocyte surface proteins that may provide new leads for studying gametocyte biology.