G-quadruplex motifs in Neisseria gonorrhoeae as anti-gonococcal targets.

Neisseria gonorrhoeae is an obligate human pathogen that causes gonorrhea and has shown a vast emergence of multidrug resistance in recent times. It is necessary to develop novel therapeutic strategies to combat this multidrug-resistant pathogen. The non-canonical stable secondary structures of nucleic acids, G-quadruplexes (GQs), are reported to regulate gene expressions in viruses, prokaryotes, and eukaryotes. Herein, we explored the whole genome of N. gonorrhoeae to mine evolutionary conserved GQ motifs. The Ng-GQs were highly enriched in the genes involved in various important biological and molecular processes of N. gonorrhoeae. Five of these GQ motifs were characterized using biophysical and biomolecular techniques. The GQ-specific ligand, BRACO-19, showed a high affinity towards these GQ motifs and stabilized them in both in vitro and in vivo conditions. The ligand showed potent anti-gonococcal activity and modulated the gene expression of the GQ-harboring genes. Strikingly, BRACO-19 also altered the biofilm formation in N. gonorrhoeae and its adhesion and invasion of the human cervical epithelial cells. In summary, the present study showed a significant role of GQ motifs in N. gonorrhoeae biology and put forward a step closer towards the search for therapeutic measures in combating the emerging antimicrobial resistance in the pathogen. KEY POINTS: •Neisseria gonorrhoeae genome is enriched in non-canonical nucleic acid structures-G-quadruplexes. •These G-quadruplexes might regulate bacterial growth, virulence, and pathogenesis. •G-quadruplex ligands inhibit biofilm formation, adhesion, and invasion of the gonococcus bacterium.

A paper microfluidic device based colorimetric sensor for the detection and discrimination of elapid versus viper envenomation.

Snake bites are a neglected tropical disease, causing mortality and severe damage to various vital organs like the nervous system, kidneys and heart. There is increasing interest in designing new antivenom treatments that are more specific to particular groups (either taxonomic or regional) of species, given the increasing evidence that current polyvalent Indian antivenom is ineffective in many situations. Under these circumstances, being able to detect the species, or a group of species, responsible for the envenomation becomes important. Unfortunately, no such diagnostic tool is available in the Indian market. Such a tool will need to be rapid, sensitive and affordable. To address this need, we have combined the power of nanotechnology and paper microfluidics and herein report a device that has the ability to detect and differentiate viper venom from elapid and scorpion venom. In principle, this assay is based on the release of the dye from the stimuli-responsive glutaraldehyde cross-linked methylene blue-loaded gelatin (GMG) nanoparticles in the presence of snake venom metalloproteases and serine proteases. The developed equipment-free assay can detect and discriminate viper venom from that of elapids and scorpions. The low-end detection limit of the sensor is ∼3.0 ng for the saw-scaled viper Echis carinatus, while the same for Russell's viper Daboia russelii is ∼6.0 ng. The performance of the sensor remains unaltered for different batches of GMG nanoparticles. Altogether, this finding establishes the role of nanotechnology and paper microfluidics in the rapid and accurate detection of viper venom.

G-Quadruplex Structures in Bacteria: Biological Relevance and Potential as an Antimicrobial Target.

DNA strands consisting of multiple runs of guanines can adopt a noncanonical, four-stranded DNA secondary structure known as G-quadruplex or G4 DNA. G4 DNA is thought to play an important role in transcriptional and translational regulation of genes, DNA replication, genome stability, and oncogene expression in eukaryotic genomes. In other organisms, including several bacterial pathogens and some plant species, the biological roles of G4 DNA and G4 RNA are starting to be explored. Recent investigations showed that G4 DNA and G4 RNA are generally conserved across plant species. In silico analyses of several bacterial genomes identified putative guanine-rich, G4 DNA-forming sequences in promoter regions. The sequences were particularly abundant in certain gene classes, suggesting that these highly diverse structures can be employed to regulate the expression of genes involved in secondary metabolite synthesis and signal transduction. Furthermore, in the pathogen Mycobacterium tuberculosis, the distribution of G4 motifs and their potential role in the regulation of gene transcription advocate for the use of G4 ligands to develop novel antitubercular therapies. In this review, we discuss the various roles of G4 structures in bacterial DNA and the application of G4 DNA as inhibitors or therapeutic agents to address bacterial pathogens.

First stellar photons for an integrated optics discrete beam combiner at the William Herschel Telescope.

We present the first on-sky results of a four-telescope integrated optics discrete beam combiner (DBC) tested at the 4.2 m William Herschel Telescope. The device consists of a four-input pupil remapper followed by a DBC and a 23-output reformatter. The whole device was written monolithically in a single alumino-borosilicate substrate using ultrafast laser inscription. The device was operated at astronomical H-band (1.6 µm), and a deformable mirror along with a microlens array was used to inject stellar photons into the device. We report the measured visibility amplitudes and closure phases obtained on Vega and Altair that are retrieved using the calibrated transfer matrix of the device. While the coherence function can be reconstructed, the on-sky results show significant dispersion from the expected values. Based on the analysis of comparable simulations, we find that such dispersion is largely caused by the limited signal-to-noise ratio of our observations. This constitutes a first step toward an improved validation of the DBC as a possible beam combination scheme for long-baseline interferometry.

G-quadruplex stabilization in the ions and maltose transporters gene inhibit Salmonella enterica growth and virulence.

The G-quadruplex structure is a highly conserved drug target for preventing infection of several human pathogens. We tried to explore G-quadruplex forming motifs as promising drug targets in the genome of Salmonella enterica that causes enteric fever in humans. Herein, we report three highly conserved G-quadruplex motifs (SE-PGQ-1, 2, and 3) in the genome of Salmonella enterica. Bioinformatics analysis inferred the presence of SE-PGQ-1 in the regulatory region of mgtA, SE-PGQ-2 in ORF of entA, and SE-PGQ-3 in the promoter region of malE and malK genes. The G-quadruplex forming sequences were confirmed by biophysical and biomolecular techniques. Cellular studies affirm the inhibitory effect of G-quadruplex specific ligands on Salmonella enterica growth. Further, PCR inhibition, reporter based assay, and RT-qPCR assays emphasize the biological relevance of G-quadruplexes in these genes. Thus, this study confirmed the presence of G-quadruplex motifs in Salmonella enterica and characterized them as a promising drug target.

Graphene Oxide Nanoparticles Modified Paper Electrode as a Biosensing Platform for Detection of the htrA Gene of O. tsutsugamushi.

The unique structural and electrochemical properties of graphene oxide (GO) make it an ideal material for the fabrication of biosensing devices. Therefore, in the present study, graphene oxide nanoparticles modified paper electrodes were used as a low-cost matrix for the development of an amperometric DNA sensor. The graphene oxide was synthesized using the modified hummers method and drop cast on a screen-printed paper electrode (SPPE) to enhance its electrochemical properties. Further, the GO/SPPE electrode was modified with a 5'NH2 labeled ssDNA probe specific to the htrA gene of Orientia tsutsugamushi using carbodiimide cross-linking chemistry. The synthesized GO was characterized using UV-Vis, FTIR, and XRD. The layer-by-layer modification of the paper electrode was monitored via FE-SEM, cyclic voltammetry, and electrochemical impedance spectroscopy (EIS). The sensor response after hybridization with single-stranded genomic DNA (ssGDNA) of O. tsutsugamushi was recorded using differential pulse voltammetry (DPV). Methylene blue (1 mM in PBS buffer, pH 7.2) was used as a hybridization indicator and [Fe(CN)6]-3/-4 (2.5 mM in PBS buffer, pH 7.2) as a redox probe during electrochemical measurements. The developed DNA sensor shows excellent sensitivity (1228.4 µA/cm2/ng) and LOD (20 pg/µL) for detection of O. tsutsugamushi GDNA using differential pulse voltammetry (DPV).

Identification and Engineering of Aptamers for Theranostic Application in Human Health and Disorders.

An aptamer is a short sequence of synthetic oligonucleotides which bind to their cognate target, specifically while maintaining similar or higher sensitivity compared to an antibody. The in-vitro selection of an aptamer, applying a conjoining approach of chemistry and molecular biology, is referred as Systematic Evolution of Ligands by Exponential enrichment (SELEX). These initial products of SELEX are further modified chemically in an attempt to make them stable in biofluid, avoiding nuclease digestion and renal clearance. While the modification is incorporated, enough care should be taken to maintain its sensitivity and specificity. These modifications and several improvisations have widened the window frame of aptamer applications that are currently not only restricted to in-vitro systems, but have also been used in molecular imaging for disease pathology and treatment. In the food industry, it has been used as sensor for detection of different diseases and fungal infections. In this review, we have discussed a brief history of its journey, along with applications where its role as a therapeutic plus diagnostic (theranostic) tool has been demonstrated. We have also highlighted the potential aptamer-mediated strategies for molecular targeting of COVID-19. Finally, the review focused on its future prospective in immunotherapy, as well as in identification of novel biomarkers in stem cells and also in single cell proteomics (scProteomics) to study intra or inter-tumor heterogeneity at the protein level. Small size, chemical synthesis, low batch variation, cost effectiveness, long shelf life and low immunogenicity provide advantages to the aptamer over the antibody. These physical and chemical properties of aptamers render them as a strong biomedical tool for theranostic purposes over the existing ones. The significance of aptamers in human health was the key finding of this review.

Chromatic response of a four-telescope integrated-optics discrete beam combiner at the astronomical L band.

We show the results of simulation and experimental study of a 4-telescope zig-zag discrete beam combiner (DBC) for long-baseline stellar interferometry working at the astronomical L band (3 - 4 µm) under the influence of a narrow bandwidth light source. Following Saviauk et al. (2013), we used a quasi-monochromatic visibility-to-pixel matrix (V2PM) for retrieving the complex coherence functions from simulated and experimentally measured power at the output of the device. Simulation and coefficient of determination (R2) measurements show that we are able to retrieve the visibility amplitudes with >95 % accuracy of our chromatic model source up to a bandwidth of 100 nm centred at 3.5 µm. We characterized a DBC manufactured by 3D ultra-fast laser inscription (ULI) written on gallium lanthanum sulphate (GLS). Experimental results showed retrieval of visibility amplitude with an accuracy of 80-90 % at 69 nm bandwidth, validating our simulation. The standard deviation of experimental phase residuals are between 0.1-0.4 rad, which shows that the retrieval procedure is sufficient to get good quality images, where phase perturbations of less than 1 rad are expected under good seeing conditions for astronomical applications.

Application of aptamers as molecular recognition elements in lateral flow assays.

Owing to their ease in operation and fast turnaround time, lateral flow assays (LFAs) are increasingly being used as point-of-care diagnostic tests for variety of analytes. In a majority of these LFAs, antibodies are used as a molecular recognition element. Antibodies have a number of limitations such as high batch-to-batch variation, poor stability, long development time, difficulty in functionalization and need for ethical approval and cold chain. All these factors pose a great challenge to scale up the antibody-based tests. In recent years, the advent of aptamer technology has made a paradigm shift in the point-of-care diagnostics owing to the various advantages of aptamers over antibodies that favour their adaptability on a variety of sensing platforms including the lateral flow. In this review, we have highlighted the advantages of aptamers over antibodies, suitability of aptamers for lateral flow platforms, different types of aptamer-based LFAs and various labels for aptamer-based LFAs. We have also provided a summary of the applications of aptamer technology in LFAs for analytical applications.

GOLD SELEX: a novel SELEX approach for the development of high-affinity aptamers against small molecules without residual activity.

GOLD SELEX, a novel SELEX approach has been developed that obviates the need for target immobilization for aptamer development. The approach purely relies on the affinity of the aptamers towards its target, to get detached from the gold nanoparticle (GNP) surface (weak attraction) after binding with its target. Thus, only the completely detached aptamers are selected for the next round of SELEX. This, in-process, also addresses the issue of residual binding and thus improves the sensitivity of the developed aptamers. As a proof of concept for establishing the utility of the approach for small molecules, we have developed aptamers against dichlorvos (DV), a pesticide in just 8 rounds. Using these aptamer candidates, we have developed an aptamer-NanoZyme (GNP having peroxidase mimic activity) based colorimetric assay. The developed aptamer displayed high affinity (Kd in sub micromolar range) and selectivity for DV. The developed assay could detect as low as 15 µM DV. The best-performing aptamer was also able to work in real samples like river water and commercial apple juice. The GOLD SELEX approach developed in this study, we believe, can act as a template for future SELEX strategy development and can replace the conventional SELEX strategy.

A novel aptamer-based test for the rapid and accurate diagnosis of pleural tuberculosis.

Pleural tuberculosis (pTB) is diagnosed by using a composite reference standard (CRS) since microbiological methods are grossly inadequate and an accurate diagnostic test remains an unmet need. The present study aimed to evaluate the utility of Mycobacterium tuberculosis (Mtb) antigen and DNA-based tests for pTB diagnosis. Patients were classified as 'Definite TB', 'Probable TB' and 'Non-TB' disease according to the CRS. We assessed the performance of in-house antigen detection assays, namely antibody-based Enzyme-Linked ImmunoSorbent Assay (ELISA) and aptamer-based Aptamer-Linked Immobilized Sorbent Assay (ALISA), targeting Mtb HspX protein and DNA-based tests namely, Xpert MTB/RIF and in-house devR-qPCR. ROC curves were generated for the combined group of 'Definite TB' and 'Probable TB' vs. 'Non-TB' disease group and cut-off values were derived to provide specificity of ≥98%. The sensitivity of ALISA was ∼93% vs. ∼24% of ELISA (p-value ≤0.0001). devR-qPCR exhibited a sensitivity of 50% vs. ∼22% of Xpert (p-value ≤0.01). This novel aptamer-based ALISA test surpasses the sensitivity criterion and matches the specificity requirement spelt out in the 'Target product profile' for extrapulmonary tuberculosis samples by Unitaid (Sensitivity ≥80%, Specificity 98%). The superior performance of the aptamer-based ALISA test indicates its translational potential to bridge the existing gap in pTB diagnosis.

Aptamer-mediated colorimetric and electrochemical detection of Pseudomonas aeruginosa utilizing peroxidase-mimic activity of gold NanoZyme.

Despite of various advancements in biosensing, a rapid, accurate, and on-site detection of a bacterial pathogen is a real challenge due to the lack of appropriate diagnostic platforms. To address this unmet need, we herein report an aptamer-mediated tunable NanoZyme sensor for the detection of Pseudomonas aeruginosa, an infectious bacterial pathogen. Our approach exploits the inherent peroxidase-like NanoZyme activity of gold nanoparticles (GNPs) in combination with high affinity and specificity of a Pseudomonas aeruginosa-specific aptamer (F23). The presence of aptamer inhibits the inherent peroxidase-like activity of GNPs by simple adsorption on to the surface of GNPs. However, in the presence of cognate target (P. aeruginosa), owing to the high affinity for P. aeruginosa, the aptamer leaves the GNP surface, allowing GNPs to resume their peroxidase-like activity, resulting in oxidation of 3,3',5,5'-tetramethylbenzidine (TMB). As TMB is an electrochemically active species, we have been able to translate the NanoZyme-based method into an ultrasensitive electrochemical assay using disposable carbon screen-printed electrode. This approach is highly sensitive and allows us to rapidly detect P. aeruginosa with a low-end detection limit of ~ 60 CFU/mL in water within 10 min. This generic aptamer-NanoZyme-based electrochemical sensing strategy may, in principle, be applicable for the detection of various other bacterial pathogens.

Evaluate the Effect of Valproate Monotherapy on the Serum Homocysteine, Folate and Vitamin B12 Levels in Epileptic Children.

BACKGROUND: The data regarding Valproate and its influence on serum folate and homocysteine levels are conflicting. The aim of this study was to evaluate whether differences exist in homocysteine, folate, and vitamin B12 levels in children receiving Valproate. METHODS: A total of 55 newly diagnosed epileptic children with ages ranging from 2 to 15 years were enrolled at the start of study but after 3 months follow up, the total sample size finally was only 50 epileptic children. 5 children dropped out of study due to poor follow up. 50 age and gender matched healthy control subjects were also studied on enrollment at the start of study. Serum homocysteine levels were analyzed by enzyme immunoassay method using the kits provided by Axis-Shield Diagnostics Ltd (Dundee DD2 1XA, United Kingdom). Serum folate and serum vitamin B12 were estimated by Competitive Chemiluminescent Enzyme Immunoassay method. RESULTS: The serum homocysteine level in epileptic children was found to be significantly increased after Valproate monotherapy as compared to before therapy. Moreover, a highly significant decrease was observed in the levels of serum folate in epileptic children after Valproate monotherapy as compared to before therapy. But a non significant difference was observed in serum vitamin B12 levels in epileptic children before and after Valproate monotherapy. CONCLUSIONS: Thus, we conclude that there is a significant increase in the levels of homocysteine and a significant decrease in the concentration of serum folate while vitamin B12 decreases non-significantly after Valproate monotherapy. The atherogenic effect of increased serum homocysteine level is well established; the patients under Valproate monotherapy should be monitored for possible atherogenic effects. Considering the above observation and results of children undergoing Valproate monotherapy, these children should be screened for levels of serum homocysteine, folate, and vitamin B12 and treated when their levels are found to be disturbed.

Aptamer-controlled reversible inhibition of gold nanozyme activity for pesticide sensing.

This study addresses the need for rapid pesticide (acetamiprid) detection by reporting a new colorimetric biosensing assay. Our approach combines the inherent peroxidase-like nanozyme activity of gold nanoparticles (GNPs) with high affinity and specificity of an acetamiprid-specific S-18 aptamer to detect this neurotoxic pesticide in a highly rapid, specific, and sensitive manner. It is shown that the nanozyme activity of GNPs can be inhibited by its surface passivation with target-specific aptamer molecules. Similar to an enzymatic competitive inhibition process, in the presence of a cognate target, these aptamer molecules leave the GNP surface in a target concentration-dependent manner, reactivating GNP nanozyme activity. This reversible inhibition of the GNP nanozyme activity can either be directly visualized in the form of color change of the peroxidase reaction product or can be quantified using UV-visible absorbance spectroscopy. This approach allowed detection of 0.1 ppm acetamiprid within an assay time of 10 min. This reversible nanozyme activation/inhibition strategy may in principle be universally applicable for the detection of a range of environmental or biomedical molecules of interest.

Characterization of G-quadruplex structures in genes involved in survival and pathogenesis of Acinetobacter baumannii as a potential drug target.

Acinetobacter baumannii is a notorious pathogen that commonly thrives in hospital environments and is responsible for numerous nosocomial infections in humans. The burgeoning multi-drug resistance leaves relatively minimal options for treating the bacterial infection, posing a significant problem and prompting the identification of new approaches for tackling the same. This motivated us to focus on non-canonical nucleic acid structures, mainly G-quadruplexes, as drug targets. G-quadruplexes have recently been gaining attention due to their involvement in multiple bacterial and viral pathogenesis. Herein, we sought to explore conserved putative G-quadruplex motifs in A. baumannii. In silico analysis revealed the presence of eight conserved motifs in genes involved in bacterial survival and pathogenesis. The biophysical and biomolecular analysis confirmed stable G-quadruplex formation by the motifs and showed a high binding affinity with the well-reported G-quadruplex binding ligand, BRACO-19. BRACO-19 exposure also decreased the growth of bacteria and downregulated the expression of G-quadruplex-harboring genes. The biofilm-forming ability of the bacteria was also affected by BRACO-19 addition. Taking all these observations into account, we have shown here for the first time the potential of G-quadruplex structures as a promising drug target in Acinetobacter baumannii, for addressing the challenges posed by this infamous pathogen.

Osteonecrosis in patients with inflammatory bowel disease: a systematic review and meta-analysis.

BACKGROUND: The relationship of inflammatory bowel disease (IBD) with osteonecrosis or avascular necrosis (AVN) is uncertain. METHODS: Systematic review to estimate the frequency of osteonecrosis in IBD was performed. Electronic databases were searched on 12 December 2022 to identify relevant studies. We planned to estimate the pooled prevalence of AVN in IBD, the risk in IBD when compared to the healthy population (without any chronic disease), and the impact of steroid use on osteonecrosis (IBD with and without steroid use). The risk of Bias was assessed with the Joanna Briggs Institute appraisal tool. RESULTS: Fifteen studies including 105 154 individuals were included. The pooled rate AVN was 10.39 per 1000 patients (95% confidence interval, 4.44-24.11, I 2  = 97%). Subgroup analysis suggested that the prevalence was lower in larger studies (>1000 participants) at 3.10, 1.07; 8.98, I 2  = 98% versus 21.03, 8.69; 50.01, I 2  = 83%. The use of steroids did not seem to increase the risk of osteonecrosis in the included studies (pooled odds ratio: 1.88, 0.55-6.41, I 2  = 39%). The systematic review was limited by the absence of comparison with the control population free of chronic disease. CONCLUSION: IBD may be associated with a risk of osteonecrosis. Future studies should assess the risk in comparison to the healthy population and the impact of disease activity and IBD therapies on the risk.

A DNA aptamer-based assay for the detection of soluble ST2, a prognostic biomarker for monitoring heart failure.

Heart failure (HF) is emerging as a leading cause of death worldwide. Estimation of BNP levels is a routine diagnosis in these patients. However, in patients having high body-mass index (BMI), renal disease or in geriatric patients, BNP level is reported to be noisy and leads to incongruous conclusion. Thus, for better risk stratification among heart failure patients, it is imperative to look for a superior biomarker. In recent times, sST2 has shown promise as a biomarker. Identifying such biomarkers in peripheral blood of HF patients, need an affine and selective molecular recognition element. Thus, in the current study an aptamer (sS9_P) against sST2 was identified from an aptamer library. Systematic Evolution of Ligands through Exponential enrichment (SELEX) derived aptamer evinced role of its primer binding domains in maintaining its selectivity. This aptamer candidate demonstrated dissociation constant (Kd) in low nanomolar range, and the Limit of Detection (LOD) was ~4 ng. Circular dichroism confirms the formation of complex stem-loop like structure. The well characterized sS9_P aptamer was used in an Aptamer Linked Immobilized Sorbent Assay (ALISA) to detect sST2 level in patients' serum (n = 99). Aptamer sS9_P has shown significant discrimination to differentiate HF patients and healthy volunteers with a reasonable specificity (~83 %) with a modest sensitivity of ~64 %. While sST-2 antibody has shown poor specificity of ~44% but good sensitivity (~87%). The insight obtained from this study indicates that a combination of aptamer and antibody-based assay can be used to design a point-of-care assay for the rapid detection of HF patients in emergency settings.

Glycohemoglobin levels with severity of periodontitis in non-diabetic population.

BACKGROUND: Periodontal disease is closely related to type 2 diabetes and is an important complication of diabetes. There are few studies about the relationship the glycohemoglobin levels with severity of periodontitis in non-diabetic population. We therefore planned this study to evaluate the glycohemoglobin levels with severity of periodontitis in non-diabetic population. METHODS: This study was conducted on 50 age and gender matched subjects in each of the three groups (according to the grades of mobility in periodontitis), a total of 150 non-diabetic periodontitis patients (Grade 1, Grade 2, and Grade 3 mobility) and 50 non-diabetic periodontitis patients with Grade 0 mobility (controls), in collaboration with the Department of Periodontics of Dental College and Department of Biochemistry, PGIMS, Rohtak, Haryana. After obtaining informed consent, fasting venous blood samples of all the non-diabetic periodontitis patients of all grades were collected aseptically for HbA1c, plasma glucose, and serum C-reactive protein (CRP) estimation. RESULTS: A total of 150 non-diabetic periodontitis patients (Grade 1, Grade 2, and Grade 3 mobility) and 50 age and gender matched controls participated in the study. There was no significant difference in fasting plasma glucose and postprandial plasma glucose in non-diabetic periodontitis patients with Grade 1, Grade 2, and Grade 3 mobility as compared to controls, non-diabetic periodontitis patients with Grade 1 mobility as compared to Grade 2, non-diabetic periodontitis patients with Grade 1 mobility as compared to Grade 3 and non-diabetic periodontitis patients with Grade 2 mobility as compared to Grade 3. Glycohemoglobin and serum C-reactive protein levels were significantly increased in non-diabetic periodontitis patients with Grade 1, Grade 2, and Grade 3 mobility as compared to controls, non-diabetic periodontitis patients with Grade 1 mobility as compared to Grade 3 and non-diabetic periodontitis patients with Grade 2 mobility as compared to Grade 3. The difference of serum C-reactive protein levels were significant. However, glycohemoglobin levels were non-significant between non-diabetic periodontitis patients with Grade 1 and Grade 2 mobility. CONCLUSIONS: The evidence of association between periodontitis and increased glycohemoglobin increases attention to the diagnosis and treatment of periodontitis, consequently improving the patient's oral health and prevention of occurrence in future diabetes. An understanding of these correlations is important to allow dental health care providers to inform patients with periodontitis of their increased risks and to counsel such patients to seek additional medical assessment or intervention as indicated.