Effector-mediated subversion of proteasome activator (PA)28αß enhances host defense against Legionella pneumophila under inflammatory and oxidative stress conditions.

Legionella pneumophila is a natural pathogen of amoebae that causes Legionnaires' Disease in immunocompromised individuals via replication within macrophages. L. pneumophila virulence and intracellular replication hinges on hundreds of Dot/Icm-translocated effector proteins, which are essential for biogenesis of the replication-permissive Legionella-containing vacuole (LCV). However, effector activity can also enhance mammalian host defense via effector-triggered immunity. The L. pneumophila effector LegC4 is important for virulence in amoebae but enhances host defense against L. pneumophila in the mouse lung and, uniquely, within macrophages activated with either tumor necrosis factor (TNF) or interferon (IFN)-γ. The mechanism by which LegC4 potentiates cytokine-mediated host defense in macrophages is unknown. Here, we found that LegC4 enhances cytokine-mediated phagolysosomal fusion with Legionella-containing vacuole (LCV) and binds host proteasome activator (PA)28α, which forms a heterooligomer with PA28ß to facilitate ubiquitin-independent proteasomal degradation of oxidant-damaged (carbonylated) proteins. We found that oxidative stress was sustained in the presence of LegC4 and that the LegC4 restriction phenotype was relieved in PA28αß-deficient macrophages and in the lungs of mice in vivo. Our data also show that oxidative stress is sufficient for LegC4-mediated restriction in macrophages producing PA28αß. PA28αß has been traditionally associated with antigen presentation; however, our data support a novel mechanism whereby effector-mediated subversion of PA28αß enhances cell-autonomous host defense against L. pneumophila under inflammatory and oxidative stress conditions. This work provides a solid foundation to evaluate induced proteasome regulators as mediators of innate immunity.

Intrabacterial Regulation of a Cytotoxic Effector by Its Cognate Metaeffector Promotes Legionella pneumophila Virulence.

Legionella pneumophila is a natural pathogen of unicellular protozoa that can opportunistically infect macrophages and cause Legionnaires' Disease. Intracellular replication is driven by hundreds of bacterial effector proteins that are translocated into infected host cells by a Dot/Icm type IV secretion system. L. pneumophila effectors are temporally regulated in part by a unique family of translocated regulatory effectors, termed metaeffectors, which bind and modulate the function of a cognate effector in host cells. Regulation of the cytotoxic effector SidI by its cognate metaeffector, MesI, is critical for L. pneumophila virulence in natural and opportunistic hosts. MesI binds and negatively regulates SidI activity in vitro, but how impaired regulation of SidI impairs L. pneumophila intracellular replication is unclear. Using a chromosomally encoded inducible expression system, we found that SidI was toxic to L. pneumophila when uncoupled from MesI. SidI enzymatic activity was required for intrabacterial toxicity since L. pneumophila growth was unaffected by induced expression of a catalytically inactive sidI allele. We also found that MesI translocation into host cells was dispensable for intracellular replication and that MesI-deficient bacteria were rapidly degraded within host cells. These data suggest that MesI promotes L. pneumophila intracellular replication by regulating SidI within the bacterium and reveal a unique role for intrabacterial effector regulation by a translocated metaeffector in L. pneumophila virulence. IMPORTANCE Legionella pneumophila replicates within phagocytic host cells using hundreds of effector protein virulence factors, which canonically subvert the function of host proteins and pathways. L. pneumophila encodes a unique family of translocated effectors called metaeffectors, which bind and regulate the function of a cognate effector in host cells. The metaeffector MesI promotes L. pneumophila virulence by regulating the cytotoxic effector SidI; however, the MesI regulatory mechanism is poorly understood. We discovered a unique intrabacterial role for MesI in L. pneumophila virulence. When uncoupled from MesI, SidI was toxic to L. pneumophila in vitro and triggered robust bacterial degradation in host cells. Furthermore, translocation of MesI was dispensable for intracellular replication, demonstrating that intrabacterial regulation of SidI contributes to L. pneumophila virulence. These data show a novel and important role for translocated effector activity within the bacterium, which challenges the dogma that L. pneumophila effectors function exclusively within host cells.

Development of robust, indigenous ELISA for detection of IgG antibodies against CoV-2 N and S proteins: mass screening.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) pandemic has adversely affected humankind and caused millions of deaths globally since January 2020. Robust and quick serological tests such as antibody detection assays for SARS-CoV-2 provide relevant information and aid in the process of vaccine development and diagnostics, as well as in sero-epidemiological monitoring of antibody response to the virus. The receptor-binding domain (RBD) of spike and nucleocapsid protein are specific targets for detecting SARS-CoV-2 antibodies. Here, we present the development of a stable spike (S) and nucleocapsid (N) protein-based ELISA antibody detection test "CoroSuchak," with 99% sensitivity, 98% specificity, cost-effective, and detection in a minimum time for serodiagnosis and mass screening of the population for antibodies against SARS-CoV-2. Blood samples were analyzed from 374 SARS-CoV-2 reverse transcription-polymerase chain reaction (RT-PCR) positive, 772 negative and asymptomatic, and 874 random groups of subjects. We found that the antibody titer was significantly higher (p < 0.0001) in infected and vaccinated group compared to the only vaccinated and only infected group. Using enzyme-linked immunosorbent assay (ELISA), we detected SARS-CoV-2 immunoglobulin G (IgG) antibodies in 118/123 (96%) infected individuals, 570/653 (87%) non-infected but vaccinated individuals, 231/237 (97%) individuals who were both infected and vaccinated, and 499/874 (57%) from randomly selected individuals from the first and second waves of the pandemic. Similarly in the third wave, 14/14 (100%) infected and 16/20 (80%) RT-PCR-negative but symptomatic subjects were detected. Thus, the highly sensitive and specific in-house developed ELISA antibody detection kit "CoroSuchak" is extremely useful to determine the seroprevalence of SARS-CoV-2 antibodies in the coronavirus-exposed population. KEY POINTS: •Indigenous kit using a combination of spike and nucleocapsid proteins and peptide sequences. •High sensitivity and specificity to detect variants. •Highly sensitive for mass screening.

Simple and facile carbon dots based electrochemical biosensor for TNF-α targeting in cancer patient's sample.

Tumour Necrosis Factor (TNF-α) is a pro-inflammatory cytokine having key roles in cell death, differentiation, survival, proliferation, migration and is a modulator of immune system. Therefore, TNF-α is an ideal biomarker for several disease diagnosis including cancer. However, out of all the biomarkers of cancer, TNF-α) is less explored for cancer detection. Only a few reports are available of developing biosensors for TNF-α targeting in human serum samples. Also, Carbon Dots (CDs) remains less explored in biosensor application. In this regard, for the first time, a sensitive and low-cost electrochemical biosensor based on CDs has developed. CDs were synthesized by simple yet facile microwave pyrolysis. Poly methyl methacrylate (PMMA) was selected as the matrix to hold CDs to fabricate the biosensing platform. This novel CD-PMMA nanocomposite featuring excellent biocompatibility, exceptional electrocatalytic conductivity, and large surface area. CD-PMMA was applied as transducing material to efficiently conjugate antibodies specific towards TNF-α and fabricate electrochemical immunosensor for specific detection of TNF-α. The fabricated immunosensor was used for the detection of TNF-α within a wide dynamic range of 0.05-160 pg mL-1 with a lower detection limit of 0.05 pg mL-1 and sensitivity of 5.56 pg mL-1 cm-2. Furthermore, this CDs based immunosensor retains high sensitivity, selectivity, and stability. This immunosensor demonstrated a high correlation with the conventional technique, Enzyme-Linked Immunosorbent Assay for early screening of cancer patient serum samples.

High-polyphenol extracts from Sorghum bicolor attenuate replication of Legionella pneumophila within RAW 264.7 macrophages.

Polyphenols derived from a variety of plants have demonstrated antimicrobial activity against diverse microbial pathogens. Legionella pneumophila is an intracellular bacterial pathogen that opportunistically causes a severe inflammatory pneumonia in humans, called Legionnaires' Disease, via replication within macrophages. Previous studies demonstrated that tea polyphenols attenuate L. pneumophila intracellular replication within mouse macrophages via increased tumor necrosis factor (TNF) production. Sorghum bicolor is a sustainable cereal crop that thrives in arid environments and is well-suited to continued production in warming climates. Sorghum polyphenols have anticancer and antioxidant properties, but their antimicrobial activity has not been evaluated. Here, we investigated the impact of sorghum polyphenols on L. pneumophila intracellular replication within RAW 264.7 mouse macrophages. Sorghum high-polyphenol extract (HPE) attenuated L. pneumophila intracellular replication in a dose-dependent manner but did not impair either bacterial replication in rich media or macrophage viability. Moreover, HPE treatment enhanced both TNF and IL-6 secretion from L. pneumophila infected macrophages. Thus, polyphenols derived from sorghum enhance macrophage restriction of L. pneumophila, likely via increased pro-inflammatory cytokine production. This work reveals commonalities between plant polyphenol-mediated antimicrobial activity and provides a foundation for future evaluation of sorghum as an antimicrobial agent.

Transcriptome analysis predicts mode of action of benzimidazole molecules against Staphylococcus aureus UAMS-1.

Antimicrobial drug resistance is one of the most critical problems that plagued the human race in modern times. Discovery of novel antibiotics is important to counter this threat. Accordingly, herein we have reported the discovery of substituted benzimidazole class of molecules with antimicrobial property (specifically against Staphylococcus aureus). They were initially identified through a random screening and a novel catalytic synthetic strategy was utilized to access them. in vitro screening and phenotypic profiling revealed the antimicrobial nature. De novo transcriptome and gene analyses predicted the putative targets. This work provides a solid foundation for developing the benzimidazoles as a target specific antimicrobial preclinical candidate.

Structure and function prediction of arsenate reductase from Deinococcus indicus DR1.

Arsenic prevalence in the environment impelled many organisms to develop resistance over the course of evolution. Tolerance to arsenic, either as the pentavalent [As(V)] form or the trivalent form [As(III)], by bacteria has been well studied in prokaryotes, and the mechanism of action is well defined. However, in the rod-shaped arsenic tolerant Deinococcus indicus DR1, the key enzyme, arsenate reductase (ArsC) has not been well studied. ArsC of D. indicus belongs to the Grx-linked prokaryotic arsenate reductase family. While it shares homology with the well-studied ArsC of Escherichia coli having a catalytic cysteine (Cys 12) and arginine triad (Arg 60, 94, and 107), the active site of D.indicus ArsC contains four residues Glu 9, Asp 53, Arg 86, and Glu 100, and with complete absence of structurally equivalent residue for crucial Cys 12. Here, we report that the mechanism of action of ArsC of D. indicus is different as a result of convergent evolution and most likely able to detoxify As(V) using a mix of positively- and negatively-charged residues in its active site, unlike the residues of E. coli. This suggests toward the possibility of an alternative mechanism of As (V) degradation in bacteria.

Dosimetric Comparison of Pencil-Beam Scanning and Photon-Based Radiation Therapy as a Boost in Carcinoma of Cervix.

PURPOSE: Brachytherapy is essential for local treatment in cervical carcinoma, but some patients are not suitable for it. Presently, for these patients, the authors prefer a boost by using intensity-modulated radiation therapy (IMRT). The authors evaluated the dosimetric comparison of proton-modulated radiation therapy versus IMRT and volumetric-modulated arc therapy (VMAT) as a boost to know whether protons can replace photons. PATIENTS AND METHODS: Five patients who received external beam radiation therapy to the pelvis by IMRT were reviewed. Three different plans were made, including pencil beam scanning (PBS), IMRT, and VMAT. The prescribed planning target volume (PTV) was 20 Gy in 4 fractions. The dose to 95% PTV (D95%), the conformity index, and the homogeneity index were evaluated for PTV. The Dmax, D2cc, and Dmean were evaluated for organs at risk along with the integral dose of normal tissue and organs at risk. RESULTS: The PTV coverage was optimal and homogeneous with modulated protons and photons. For PBS, coverage D95% was 20.01 ± 0.02 Gy (IMRT, 20.08 ± 0.06 Gy; VMAT, 20.1 ± 0.04 Gy). For the organs at risk, Dmax of the bladder for PBS was 21.05 ± 0.05 Gy (IMRT, 20.8 ± 0.21 Gy; VMAT, 21.65 ± 0.41 Gy) while the Dmax for the rectum for PBS was 21.04 ± 0.03 Gy (IMRT, 20.81 ± 0.12 Gy; VMAT, 21.66 ± 0.38 Gy). Integral dose to normal tissues in PBS was 14.17 ± 2.65 Gy (IMRT, 25.29 ± 6.35 Gy; VMAT, 25.24 ± 6.24 Gy). CONCLUSIONS: Compared with photons, modulated protons provide comparable conformal plans. However, PBS reduces the integral dose to critical structures significantly compared with IMRT and VMAT. Although PBS may be a better alternative for such cases, further research is required to substantiate such findings.

Dosimetric influence of photon beam energy and number of arcs on volumetric modulated arc therapy in carcinoma cervix: A planning study.

AIM: Aim of the present study was to compare the dosimetric impact of different photon beam energies and number of arcs in the treatment of carcinoma cervix. BACKGROUND: Carcinoma cervix is a common cancer in women worldwide with a high morbidity rate. Radiotherapy is used to treat such tumours. Volumetric Modulated Arc Therapy (VMAT) is considered superior to other techniques with multiple arcs and energies. MATERIALS AND METHODS: Twenty patients with carcinoma cervix underwent radiotherapy in a prospective observation study conducted at our institute. Volumetric modulated arc plans with 6 MV, 10 MV and 15 MV photon energies using single arc (SA) and dual arc (DA) were generated. Several physical indices for planning target volume (PTV) like V95%, V100%, V110%, D98%, D50%, D2% and total number of MUs were compared. Normal Tissue Integral Dose (NTID) and dose to a shell structure PHY2.5 and PHY5.0 were analyzed. RESULTS: Comparable dose coverage to PTV was observed for all the energies and arcs. CI for DA6MV (1.095) was better than SA6MV (1.127), SA10MV (1.116) and SA15MV (1.116). Evaluated parameters showed significant reduction in OAR doses. Mean bladder dose for DA6MV (41.90 Gy) was better than SA6MV (42.48 Gy), SA10MV (42.08 Gy) and SA15MV (41.93 Gy). Similarly, p-value for the mean rectal dose calculated was 0.001 (SA6 vs 15), 0.013 (DA6 vs 10) and 0.003 (DA6 vs 15) and subsequently favoured DA6MV. Difference in NTID was very small. CONCLUSIONS: The study showed no greater advantage of higher energy, and DA VMAT plan with 6 MV photon energy was a good choice of treatment for carcinoma cervix as it delivered a highly homogeneous and conformal plan with superior target coverage and better OAR sparing.

Comparison between weekly cisplatin-enhanced radiotherapy and cetuximab-enhanced radiotherapy in locally advanced head and neck cancer: first retrospective study in Asian population.

AIM: To present a direct comparison between chemotherapy-enhanced radiotherapy (CERT) and biotherapy-enhanced radiotherapy (BERT) in locally advanced head and neck cancer. METHODS: It is a retrospective analysis of 53 patients with locally advanced head and neck cancer treated from August 2006 to December 2008. For CERT, patients received weekly cisplatin (40 mg/m2 ) and for BERT, a loading dose of 400 mg/m2 of cetuximab given one week prior to radiotherapy followed by 250 mg/m2 given weekly along with radiotherapy. Disease-free survival (DFS) and overall survival (OS) were computed with Kaplan-Meier curve with log-rank test for comparison between the two groups. Multivariate Cox proportional hazards regression analysis was performed to estimate the impact of known relevant prognostic factors on DFS and OS. RESULTS: The median DFS was significantly better with CERT than BERT group (50.82 vs 11.66 months; P = 0.031). The 3 years DFS was significantly higher in CERT group than in BERT group (60.0% vs 14.3%; P = 0.022). The median OS was significantly better with CERT than BERT group (53.61 vs 32.55 months; P = 0.044). The 3 years OS was also significantly higher in CERT group than in BERT group (74.0% vs 42.1%; P = 0.032). There were no significant differences in acute toxicities of all grade and grade ≥3 between the two groups. The compliance to treatment and assisted feeding dependency for more than 6 months duration were also not significantly different. CONCLUSION: CERT is associated with better outcome with no significantly increased acute toxicities compared to BERT.

Tumour volumes: Predictors of early treatment response in locally advanced head and neck cancers treated with definitive chemoradiation.

AIM: To analyse and predict early response 3 months post definitive chemoradiation (CCRT) utilising tumour volume (TV) measurement in locally advanced head and neck cancers (LAHNC). BACKGROUND: LAHNC are 3-dimentional lesions. The largest diameter of these tumours measured for T-classification may not necessarily reflect the true tumour dimensions. TV accurately reflects the tumour burden because it is a measurement of tumour burden in all three dimensions. MATERIALS AND METHODS: It is a single institutional prospective study including 101 patients with LAHNC treated with definitive CCRT. TV data noted were primary tumour volume (PTV), total nodal volume (TNV) and total tumour volume (TTV). Response evaluation was done at 3 months after the completion of definitive CCRT and patients were categorised either having achieved complete response (CR) or residual disease. RESULTS: Patients who had not achieved CR were found to have larger TV compared with those who had achieved CR. There were significant inverse correlations between PTV and response (median 16.37 cm(3) vs. 45.2 cm(3); p = 0.001), and between TTV and response (median 36.14 cm(3) vs. 66.06 cm(3); p < 0.001). Receiver operating characteristic (ROC) analysis identified an "optimal cut-off" value of 41 cm(3) for PTV and 42 cm(3) for TTV above and below which the magnitude of difference in response was the greatest. CONCLUSIONS: If response evaluation 3 months post CCRT is to be predicted it is simply not enough to measure the largest single dimension of the tumour. TV seems to be a better and more accurate reflection of the true total tumour burden or extent of the disease.

Radiation-induced dental caries, prevention and treatment - A systematic review.

Treatment of head and neck cancers (HNCs) involves radiotherapy. Patients undergoing radiotherapy for HNCs are prone to dental complications. Radiotherapy to the head and neck region causes xerostomia and salivary gland dysfunction which dramatically increases the risk of dental caries and its sequelae. Radiation therapy (RT) also affects the dental hard tissues increasing their susceptibility to demineralization following RT. Postradiation caries is a rapidly progressing and highly destructive type of dental caries. Radiation-related caries and other dental hard tissue changes can appear within the first 3 months following RT. Hence, every effort should be focused on prevention to manage patients with severe caries. This can be accomplished through good preoperative dental treatment, frequent dental evaluation and treatment after RT (with the exception of extractions), and consistent home care that includes self-applied fluoride. Restorative management of radiation caries can be challenging. The restorative dentist must consider the altered dental substrate and a hostile oral environment when selecting restorative materials. Radiation-induced changes in enamel and dentine may compromise bonding of adhesive materials. Consequently, glass ionomer cements have proved to be a better alternative to composite resins in irradiated patients. Counseling of patients before and after radiotherapy can be done to make them aware of the complications of radiotherapy and thus can help in preventing them.

Improving the accuracy of target volume delineation by combined use of computed tomography, magnetic resonance imaging and positron emission tomography in head and neck carcinomas.

OBJECTIVE: Conformal radiation therapy mandates accurate delineation of target volumes, which requires incorporation of modern imaging modalities like magnetic resonance imaging (MRI) and positron emission tomography (PET) in addition to conventionally used computed tomography (CT). This can resolve discrepancies in target delineation in head and neck carcinomas resulting in better local control. We hereby report the comparison of Gross Tumor Volumes (GTVs) (primary) drawn using PET, CT and MRI and their concordance indices. METHODS AND MATERIAL: Twenty five patients with head and neck cancer were taken into this study. MRI, PET and CT planning scans were done as per standard guidelines. Three sets of primary GTVs namely GTV- PET, GTV-CT and GTV-MRI were contoured on fused images. All the three volumes and concordances among the volumes were analyzed. RESULT: The mean GTV-CT, GTV-PET and GTV-MRI volumes were 29.65 cc ± 31.27, 32.05 cc ± 33.75 and 24.85 cc ± 25.28 respectively. There was a significant difference in the GTV-MRI & GTV-CT volumes (P = 0.023) and GTV-PET & GTV-MRI volumes (P = 0.049). However, there was no significant difference in the GTV-PET & GTV-CT volume (P = 0.468). The mean CI (PET-MRI), CI (CT-MRI) and CI (PET-CT) was 0.42, 0.46 and 0.47 respectively, which depicts a moderate concordance. CONCLUSION: PET and MRI are useful imaging tools in head and neck malignancies and should be used in conjunction with CT scan for improved target volume delineation.

Is volumetric modulated arc therapy (RapidArc) better than intensity modulated radiotherapy for gynecological malignancies? A dosimetric comparison.

AIMS: Evaluation of the dosimetric advantage, if any, of RapidArc (double arc and triple arc [DA and TA]) over standard fixed field intensity-modulated radiation therapy (IMRT) in gynecologic malignancies. MATERIALS AND METHODS: A total of 20 cases of gynecologic malignancies were included. Static IMRT sliding window, single arc (SA), DA and TA plans were generated with eclipse planning system. The prescribed dose was 50.4 Gy/28# to the planning target volumes. RESULTS: IMRT provided target coverage equivalent to DA and inferior to TA (D95% [in Gy]--49.94, 49.58, 49.96, 50.17 for IMRT, SA, DA and TA respectively--all observations in the same sequence). Conformity index 90 (CI 90 (0.964, 0.927, 0.918, 0.822) and homogeneity index (0.0683, 0.119, 0.098, 0.097) of IMRT were superior. TA was superior to other arcs in all parameters except CI 90 (P=0.805) and bladder dose (lower in DA). Rectal, bladder and bowel sparing was best achieved with IMRT followed by TA; bilateral femur dose was lower in arcs. The total monitor units and treatment time of arcs were significantly lower than IMRT, reduced by a factor of 2.41-2.59 and 3.2-3.5 respectively (All P values significant). CONCLUSION: IMRT provided better overall plan for gynecologic malignancies with lower organs at risk dose and target coverage equivalent to DA and TA. Treatment delivery efficiency was higher with RapidArc. The TA plan is dosimetrically superior to DA, but the gain is small. The decision whether or not to add a third arc for a small gain should be individualized.

Chemoradiotherapy in the management of locally advanced squamous cell carcinoma esophagus: is surgical resection required?

PURPOSE: The present study aims to evaluate benefit of adding surgery to chemoradiotherapy alone in management of carcinoma esophagus. METHODS: We retrospectively analyzed 45 eligible patients of squamous cell esophageal carcinoma which were enrolled from February 2008 to April 2009. All patients were treated with chemoradiotherapy (50.40 Gy with 40 mg/m(2) of weekly cisplatin). Tumor response was assessed after 6 weeks of treatment. Patients with resectable disease were subjected to surgical resection (arm A) and remaining was kept on regular clinical follow-up (arm B). Overall survival (OS) was selected as the primary endpoint. The secondary end points were disease-free survival (DFS) and clinical toxicities. RESULTS: Median follow-up was 13.6 months. Pathological complete response was seen in 60.9 % patients in arm A. In arm B, 77.3 % patients attained radiological complete response (p = 0.194). The median OS was 16.4 and 19.1 months (p = 0.388) and median DFS was 5.8 and 4.1 months (p = 0.347) in arm A and B, respectively. The 2-year survival probability was 39.1 and 36.4 % (p = 0.387) in arm A and B, respectively. The recurrence probability was 56.5 % (SE = 5.6 %) and 45.5 % (SE = 4.2 %) (p = 0.328) in arm A and B, respectively. The probability of loco regional recurrence was more in arm B than in arm A (p = 0.002). CONCLUSIONS: The study suggests that there is no difference in clinical toxicity profiles or survival outcomes with either definitive chemoradiotherapy or chemoradiation followed by surgery in management of locally advanced esophageal cancer.