Facemask analyses for the non-invasive detection of chronic and acute P. aeruginosa lung infections using nanoparticle-based immunoassays.

Pseudomonas aeruginosa (P. aeruginosa) is a pathogen that persistently colonizes the respiratory tract of patients with chronic lung diseases. The risk of acquiring a chronic P. aeruginosa infection can be minimized by rapidly detecting the pathogen in the patient's airways and promptly administrating adequate antibiotics. However, the rapid detection of P. aeruginosa in the lungs involves the analysis of sputum, which is a highly complex matrix that is not always available. Here, we propose an alternative diagnosis based on analyzing breath aerosols. In this approach, nanoparticle immunosensors identify bacteria adhered to the polypropylene layer of a surgical facemask that was previously worn by the patient. A polypropylene processing protocol was optimized to ensure the efficient capture and analysis of the target pathogen. The proposed analytical platform has a theoretical limit of detection of 105 CFU mL-1 in aerosolized mock samples, and a dynamic range between 105 and 108 CFU mL-1. When tested with facemasks worn by patients, the biosensors were able to detect chronic and acute P. aeruginosa lung infections, and to differentiate them from respiratory infections caused by other pathogens. The results shown here pave the way to diagnose Pseudomonas infections at the bedside, as well as to identify the progress from chronic to acute infection.

A paper biosensor for overcoming matrix effects interfering with the detection of sputum pyocyanin with competitive immunoassays.

Detecting sputum pyocyanin (PYO) with a competitive immunoassay is a promising approach for diagnosing Pseudomonas aeruginosa respiratory infections. However, it is not possible to perform a negative control to evaluate matrix-effects in competitive immunoassays, and the highly complex sputum matrix often interferes with target detection. Here, we show that these issues are alleviated by performing competitive immunoassays with a paper biosensor. The biosensing platform consists of a paper reservoir, which contains antibody-coated gold nanoparticles, and a substrate containing a competing recognition element, which is a piece of paper modified with an albumin-antigen conjugate. Detection of PYO with a limit of detection of 4.7·10-3 µM and a dynamic range between 4.7·10-1 µM and 47.6 µM is accomplished by adding the sample to the substrate with the competing element and pressing the reservoir against it for 5 min. When tested with patient samples, the biosensor was able to qualitatively differentiate spiked from non-spiked samples, whereas ELISA did not show a clear cut-off between them. Furthermore, the relative standard deviation was lower when determining sputum with the paper-based biosensor. These features, along with a mild liquefaction step that circumvents the use of harsh chemicals or instruments, make our biosensor a good candidate for diagnosing Pseudomonas infections at the bedside through the detection of sputum PYO.

Medial gastrocnemius rotational flap for the reconstruction of extensor mechanism disruption in periprosthetic knee infections: a retrospective series with minimum two year follow-up.

PURPOSE: Extensor mechanism disruption following total knee arthroplasty has a prevalence ranging from 0.3 to 3%. Its management is challenging, especially in case of associated infection of the prosthetic implant. Surgical options are limited due to the septic process, and the use of allograft or synthetic mesh are not recommended. The aim of this study was to report clinical outcomes, complications, survival, and surgical technique of medial gastrocnemius flap for the treatment of extensor mechanism disruptions associated with periprosthetic knee infection. METHODS: This is a retrospective study from a prospectively collected arthroplasty registry from 2012 to 2019. Patients who received the gastrocnemius flap in the setting of a two-stage knee replacement for periprosthetic infection were included. Results of physical examination, Knee Society Score, Oxford Knee Score, and measurement of the range of motion registered pre-operatively were compared to those obtained at last follow-up. Survival was analysed through Kaplan-Meier curve. RESULTS: A total of 15 patients were included, with a mean age of 63.4 years (range 36-77). The reconstruction of the extensor mechanism demonstrated a success rate of 73.3%. The mean extension lag at final follow-up was 7.5° (range, 0-30). The mean Knee Society Score and Oxford Knee Score improved from 29.0 (range, 21-36) and 17.5 (range, 13-22) respectively, to 82.9 (range, 74-89) and 36.0 (range, 33-39). CONCLUSION: Medial gastrocnemius rotational flap is a reliable option for joint and limb salvage in case of periprosthetic knee infection associated with wide soft tissue degeneration and extensor mechanism disruption. The technique and surgical protocol presented in this study are reproducible and guaranteed good clinical outcomes and infection control.

Spacer exchange in persistent periprosthetic joint infection: microbiological evaluation and survivorship analysis.

PURPOSE: The purposes of this study were to determine demographics and characteristics of patients who underwent spacer exchange for persistent infection in the setting of two-stage arthroplasty for periprosthetic joint infection, to describe the microbiology of pathogens involved, to analyze survivorship free from infection in these patients. METHODS: The institutional prospectively collected database was reviewed to enroll patients with minimum 2 years follow-up. Patients who underwent two-stage procedure for septic arthritis were excluded, as were patients who had spacer fracture or dislocation. RESULTS: A total of 34 patients (41 procedures) were included. Mean age was 65.0 ± 12.8 years. Mean follow-up was 53.4 ± 24.8 months. Mean number of previous procedures was 3.6 ± 1.2. A total of 27 (79.4%) patients underwent final reimplantation. The most frequently isolated pathogen in spacer exchange was Staphylococcus epidermidis (10 cases, 28.6%). Polymicrobial cultures were obtained from 9 (25.71%) patients, 10 (28.6%) presented culture-negative infections. A total of 11 (32.4%) resistant pathogens were isolated, and 16 (47.0%) difficult to treat pathogens were detected. Eradication rate was 78.8%. Overall survivorship of implants after final reimplantation was 72.8% at 51.8 months. CONCLUSION: Surgeons should be aware that subjects necessitating spacer exchange often present multiple comorbidities, previous staged revision failures, soft-tissue impairment and difficult to treat infection. In these patients, spacer exchange provides good clinical results and infection eradication, preventing arthrodesis or amputation.

Rapid Identification and Classification of Pathogens That Produce Carbapenemases and Cephalosporinases with a Colorimetric Paper-Based Multisensor.

Infections caused by bacteria that produce ß-lactamases (BLs) are a major problem in hospital settings. The phenotypic detection of these bacterial strains requires culturing samples prior to analysis. This procedure may take up to 72 h, and therefore it cannot be used to guide the administration of the first antibiotic regimen. Here, we propose a multisensor for identifying pathogens bearing different types of ß-lactamases above the infectious dose threshold within 90 min that does not require culturing samples. Instead, bacterial cells are preconcentrated in the cellulose scaffold of a paper-based multisensor. Then, 12 assays are performed in parallel to identify whether the pathogens produce carbapenemases and/or cephalosporinases, including metallo-ß-lactamases, extended-spectrum ß-lactamases (ESBLs), and AmpC enzymes. The multisensor generates an array of colored spots that can be quantified with image processing software and whose interpretation leads to the detection of the different enzymes depending on their specificity toward the hydrolysis of certain antibiotics, and/or their pattern of inhibition or cofactor activation. The test was validated for the diagnosis of urinary tract infections. The inexpensive paper platform along with the uncomplicated colorimetric readout makes the proposed prototypes promising for developing fully automated platforms for streamlined clinical diagnosis.

Improved cytometric analysis of untouched lung leukocytes by enzymatic liquefaction of sputum samples.

BACKGROUND: Phenotyping sputum-resident leukocytes and evaluating their functional status are essential analyses for exploring the cellular basis of pathological processes in the lungs, and flow cytometry is widely recognized as the gold-standard technique to address them. However, sputum-resident leukocytes are found in respiratory samples which need to be liquefied prior to cytometric analysis. Traditional liquefying procedures involve the use of a reducing agent such as dithiothreitol (DTT) in temperature-controlled conditions, which does not homogenize respiratory samples efficiently and impairs cell viability and functionality. METHODS: Here we propose an enzymatic method that rapidly liquefies samples by means of generating O2 bubbles with endogenous catalase. Sputum specimens from patients with suspected pulmonary infection were treated with DTT, the enzymatic method or PBS. We used turbidimetry to compare the liquefaction degree and cell counts were determined using a hemocytometer. Finally, we conducted a comparative flow cytometry study for evaluating frequencies of sputum-resident neutrophils, eosinophils and lymphocytes and their activation status after liquefaction. RESULTS: Enzymatically treated samples were better liquefied than those treated with DTT or PBS, which resulted in a more accurate cytometric analysis. Frequencies of all cell subsets analyzed within liquefied samples were comparable between liquefaction methods. However, the gentle cell handling rendered by the enzymatic method improves cell viability and retains in vivo functional characteristics of sputum-resident leukocytes (with regard to HLA-DR, CD63 and CD11b expression). CONCLUSION: In conclusion, the proposed enzymatic liquefaction method improves the cytometric analysis of respiratory samples and leaves the cells widely untouched for properly addressing functional analysis of lung leukocytes.

Plasma-induced nanoparticle aggregation for stratifying COVID-19 patients according to disease severity.

Stratifying patients according to disease severity has been a major hurdle during the COVID-19 pandemic. This usually requires evaluating the levels of several biomarkers, which may be cumbersome when rapid decisions are required. In this manuscript we show that a single nanoparticle aggregation test can be used to distinguish patients that require intensive care from those that have already been discharged from the intensive care unit (ICU). It consists of diluting a platelet-free plasma sample and then adding gold nanoparticles. The nanoparticles aggregate to a larger extent when the samples are obtained from a patient in the ICU. This changes the color of the colloidal suspension, which can be evaluated by measuring the pixel intensity of a photograph. Although the exact factor or combination of factors behind the different aggregation behavior is unknown, control experiments demonstrate that the presence of proteins in the samples is crucial for the test to work. Principal component analysis demonstrates that the test result is highly correlated to biomarkers of prognosis and inflammation that are commonly used to evaluate the severity of COVID-19 patients. The results shown here pave the way to develop nanoparticle aggregation assays that classify COVID-19 patients according to disease severity, which could be useful to de-escalate care safely and make a better use of hospital resources.

Improving the Quantification of Colorimetric Signals in Paper-Based Immunosensors with an Open-Source Reader.

Measuring the colorimetric signals produced by the biospecific accumulation of colorimetric probes and recording the results is a key feature for next-generation paper-based rapid tests. Manual processing of these tests is time-consuming and prone to a loss of accuracy when interpreting faint and patchy signals. Proprietary, closed-source readers and software companies offering automated smartphone-based assay readings have both been criticized for interoperability issues. Here, we introduce a minimal reader prototype composed of open-source hardware and open-source software that has the benefits of automatic assay quantification while avoiding the interoperability issues associated with closed-source readers. An image-processing algorithm was developed to automate the selection of an optimal region of interest and measure the average pixel intensity. When used to quantify signals produced by lateral flow immunoassays for detecting antibodies against SARS-CoV-2, results obtained with the proposed algorithm were comparable to those obtained with a manual method but with the advantage of improving the precision and accuracy when quantifying small spots or faint and patchy signals.

Clinical radiographic outcomes and survivorship of medial pivot design total knee arthroplasty: a systematic review of the literature.

BACKGROUND: Total knee arthroplasty is a reliable procedure able to reduce pain and disability in patients suffering from osteoarthritis. However, a considerable percentage of patients still experiences unsatisfactory results. Medial pivot total knee arthroplasty has been introduced in the clinical practice to overcome problems related with classic design implants and better mimic native knee kinematics. The aim of this study was to analyze survivorship and clinical and radiographic outcomes of medial pivot implants. METHODS: A systematic research was conducted in eight different databases. Thirty-four studies met the inclusion criteria and were included in the analysis. Data on objective and patients-reported outcomes, radiographic alignment, and survivorship were collected and analyzed. Revision rate was expressed as revision per 100 components years. RESULT: A total of 3377 procedures were included. Mean follow-up was 85.7 months (range, 12-182). The revision per 100 components years was 0.19, which corresponds to a revision rate of 1.9% after 10 years. Mean post-operative range of motion was 117.3 ± 0.4°. Mean clinical and functional Knee Society Score were, respectively, 85.9 ± 1.1 and 84.7 ± 3.5 at final follow-up. Post-operative femorotibial alignment was 177.1 ± 0.5°. Alfa and beta angles were 95.7 ± 0.1° and 89.2 ± 0.1°, respectively. Gamma and delta angles were 2.3 ± 0.6° and 86.7 ± 0.4°. CONCLUSION: Medial pivoting implants provided excellent survivorship and low revision rate, as well as good-to-excellent results in term of objective and patient-reported clinical outcomes, and reliable correction of radiographic parameters. More high-quality studies with long-term follow-up are needed to clarify the role of medial pivoting implants.

Immunodetection of Lung IgG and IgM Antibodies against SARS-CoV-2 via Enzymatic Liquefaction of Respiratory Samples from COVID-19 Patients.

Lung-secreted IgG and IgM antibodies are valuable biomarkers for monitoring the local immune response against respiratory infections. These biomarkers are found in lower airway secretions that need to be liquefied prior to analysis. Traditional methods for sample liquefaction rely on reducing disulfide bonds, which may damage the structure of the biomarkers and hamper their immunodetection. Here, we propose an alternative enzymatic method that uses O2 bubbles generated by endogenous catalase enzymes in order to liquefy respiratory samples. The proposed method is more efficient for liquefying medium- and high-viscosity samples and does not fragment the antibodies. This prevents damage to antigen recognition domains and recognition sites for secondary antibodies that can decrease the signal of immunodetection techniques. The suitability of the enzymatic method for detecting antibodies in respiratory samples is demonstrated by detecting anti-SARS-CoV-2 IgG and IgM to viral N-protein with gold standard ELISA in bronchial aspirate specimens from a multicenter cohort of 44 COVID-19 patients. The enzymatic detection sharply increases the sensitivity toward IgG and IgM detection compared to the traditional approach based on liquefying samples with dithiothreitol. This improved performance could reveal new mechanisms of the early local immune response against respiratory infections that may have gone unnoticed with current sample treatment methods.

Mobile origami immunosensors for the rapid detection of urinary tract infections.

Urinary tract infections (UTI) have a high prevalence and can yield poor patient outcomes if they progress to urosepsis. Here we introduce mobile origami biosensors that detect UTIs caused by E. coli at the bedside in less than 7 minutes. The origami biosensors are made of a single piece of paper that contains antibody-decorated nanoparticles. When the urine sample contains E. coli, the biosensors generate colored spots on the paper strip. These are then quantified with a mobile app that calculates the pixel intensity in real time. The tests are highly specific and do not cross-react with other common uropathogens. Furthermore, the biosensors only yielded one false negative result when queried with a panel containing 57 urine samples from patients, which demonstrates that they have excellent sensitivity and specificity. This, along with the rapid assay time and smartphone-based detection, makes them useful for aiding in the diagnosis of UTIs at the point of care.

Optimized detection of lung IL-6 via enzymatic liquefaction of low respiratory tract samples: application for managing ventilated patients.

Lung IL-6 is a promising biomarker for predicting respiratory failure during pulmonary infections. This biomarker is found in respiratory samples which need to be liquefied prior to analysis. Traditional liquefying methods use reducing agents such as dithiothreitol (DTT). However, DTT impairs immunodetection and does not liquefy highly viscous samples. We propose an enzymatic method that liquefies samples by means of generating O2 bubbles with endogenous catalase. Low respiratory tract specimens from 48 mechanically ventilated patients (38 with SARS-CoV-2 infection) were treated with DTT or with the enzymatic method. We used turbidimetry to compare the liquefaction degree and IL-6 was quantified with ELISA. Finally, we used AUC-ROC, time-to-event and principal component analysis to evaluate the association between respiratory compromise or local inflammation and IL-6 determined with both methods. Enzymatically treated samples were better liquefied than those reduced by DTT, which resulted in higher ELISA signals. Lung IL-6 levels obtained with the enzymatic procedure were negatively correlated with the oxygenation index (PaO2/FiO2) and the time of mechanical ventilation. The proposed enzymatic liquefaction method improves the sensitivity for lung IL-6 detection in respiratory samples, which increases its predictive power as a biomarker for evaluating respiratory compliance.

Paper biosensors for detecting elevated IL-6 levels in blood and respiratory samples from COVID-19 patients.

Decentralizing COVID-19 care reduces contagions and affords a better use of hospital resources. We introduce biosensors aimed at detecting severe cases of COVID-19 in decentralized healthcare settings. They consist of a paper immunosensor interfaced with a smartphone. The immunosensors have been designed to generate intense colorimetric signals when the sample contains ultralow concentrations of IL-6, which has been proposed as a prognosis biomarker of COVID-19. This is achieved by combining a paper-based signal amplification mechanism with polymer-filled reservoirs for dispensing antibody-decorated nanoparticles and a bespoken app for color quantification. With this design we achieved a low limit of detection (LOD) of 10-3 pg mL-1 and semi-quantitative measurements in a wide dynamic range between 10-3 and 102 pg mL-1 in PBS. The assay time is under 10 min. The low LOD allowed us to dilute blood samples and detect IL-6 with an LOD of 1.3 pg mL-1 and a dynamic range up to 102 pg mL-1. Following this protocol, we were able to stratify COVID-19 patients according to different blood levels of IL-6. We also report on the detection of IL-6 in respiratory samples (bronchial aspirate, BAS) from COVID-19 patients. The test could be easily adapted to detect other cytokines such as TNF-α and IL-8 by changing the antibodies decorating the nanoparticles accordingly. The ability of detecting cytokines in blood and respiratory samples paves the way for monitoring local inflammation in the lungs as well as systemic inflammation levels in the body.

Nanoparticle transfer biosensors for the non-invasive detection of SARS-CoV-2 antigens trapped in surgical face masks.

Detecting SARS-CoV-2 antigens in respiratory tract samples has become a widespread method for screening new SARS-CoV-2 infections. This requires a nasopharyngeal swab performed by a trained healthcare worker, which puts strain on saturated healthcare services. In this manuscript we describe a new approach for non-invasive COVID-19 diagnosis. It consists of using mobile biosensors for detecting viral antigens trapped in surgical face masks worn by patients. The biosensors are made of filter paper containing a nanoparticle reservoir. The nanoparticles transfer from the biosensor to the mask on contact, where they generate colorimetric signals that are quantified with a smartphone app. Sample collection requires wearing a surgical mask for 30 min, and the total assay time is shorter than 10 min. When tested in a cohort of 27 patients with mild or no symptoms, an area under the receiving operating curve (AUROC) of 0.99 was obtained (96.2 % sensitivity and 100 % specificity). Serial measurements revealed a high sensitivity and specificity when masks were worn up to 6 days after diagnosis. Surgical face masks are inexpensive and widely available, which makes this approach easy to implement anywhere. The excellent sensitivity, even when tested with asymptomatic patient samples, along with the mobile detection scheme and non-invasive sampling procedure, makes this biosensor design ideal for mass screening.

Clinical outcomes and survivorship of two-stage total hip or knee arthroplasty in septic arthritis: a retrospective analysis with a minimum five-year follow-up.

PURPOSE: Septic arthritis of the native joint is challenging for orthopedic surgeons because it may lead to wide bone defects and severe impairment of joint function. This study aimed to analyze clinical functional outcomes, the rate of infection eradication, and survival of implants of patients who underwent two-stage arthroplasty for septic arthritis of the hip and knee. METHODS: A retrospective single-centre analysis was conducted of patients treated for septic arthritis of the hip and knee joints through a two-stage surgery between 2012 and 2015. Clinical and radiological records were gathered from the prospectively collected Institutional Arthroplasty Registry. Patients' pre-operative Harris hip scores and Knee Society scores were compared with those obtained at the latest follow-up. Kaplan-Meier curves were generated to assess survival of implants. RESULTS: Forty-seven patients were included. The mean follow-up was 85.2 ± 15.4 months. The Harris hip score improved from 39.4 ± 9.9 to 84.5 ± 10.8 points (p < 0.001). The Knee Society score improved from 40.7 ± 8.4 to 86.0 ± 7.8 points (p < 0.001). Knee Society score-function increased from 25.7 ± 14.2 to 85.4 ± 23.4 points (p < 0.001). The infection eradication rates were 92.0% and 90.9% in patients who underwent hip and knee operation, respectively (p = 0.891). Overall survivorship of implants after the second stage was 93.6%. CONCLUSIONS: Two-stage arthroplasty provides good to excellent clinical outcomes in cases of active septic arthritis of the hip and the knee, high rates of infection control, and implant survival.

Intralesional ultrasound-guided combined treatment with triamcinolone plus lincomycin in hidradenitis suppurativa: A pilot study.

Hidradenitis suppurativa (HS) is a chronic, inflammatory, disease of the hair follicle. Intralesional corticosteroid treatment in HS patients has been reported, and while several data described this route of administration as an efficient delivery system, its efficacy is still debated. The aim of this study was to explore the clinical efficacy and the effect on quality of life (QoL) of an innovative intralesional treatment in HS patients. This was an interventional prospective study. The treatment consisted of two intralesional ultrasound-guided injections of triamcinolone plus lincomycin, at baseline and after 2 weeks. Lesions and QoL were evaluated at baseline and at 4 weeks following intralesional therapy. All clinical variables of 36 HS patients significantly improved after 4 weeks. Mean values of the visual analog scale (VAS) pain decreased from 4.6 to 1.5, P = .027. The Bodily Pain (BP) scale of the Short-Form Health Survey (SF-36) significantly improved from 36.2 at baseline to 53.9 at 4-week follow-up (P < .001). On a scale from 0 to 10, over 90% of the patients gave a satisfaction score of 8 or more. This combination of corticosteroids and antibiotics delivered intralesionally seems to be effective, as it improved both patient- and physician-reported outcomes.

Loss of M1 Receptor Dependent Cholinergic Excitation Contributes to mPFC Deactivation in Neuropathic Pain.

In chronic pain, the medial prefrontal cortex (mPFC) is deactivated and mPFC-dependent tasks such as attention and working memory are impaired. We investigated the mechanisms of mPFC deactivation in the rat spared nerve injury (SNI) model of neuropathic pain. Patch-clamp recordings in acute slices showed that, 1 week after the nerve injury, cholinergic modulation of layer 5 (L5) pyramidal neurons was severely impaired. In cells from sham-operated animals, focal application of acetylcholine induced a left shift of the input/output curve and persistent firing. Both of these effects were almost completely abolished in cells from SNI-operated rats. The cause of this impairment was an ∼60% reduction of an M1-coupled, pirenzepine-sensitive depolarizing current, which appeared to be, at least in part, the consequence of M1 receptor internalization. Although no changes were detected in total M1 protein or transcript, both the fraction of the M1 receptor in the synaptic plasma membrane and the biotinylated M1 protein associated with the total plasma membrane were decreased in L5 mPFC of SNI rats. The loss of excitatory cholinergic modulation may play a critical role in mPFC deactivation in neuropathic pain and underlie the mPFC-specific cognitive deficits that are comorbid with neuropathic pain.SIGNIFICANCE STATEMENT The medial prefrontal cortex (mPFC) undergoes major reorganization in chronic pain. Deactivation of mPFC output is causally correlated with both the cognitive and the sensory component of neuropathic pain. Here, we show that cholinergic excitation of commissural layer 5 mPFC pyramidal neurons is abolished in neuropathic pain rats due to a severe reduction of a muscarinic depolarizing current and M1 receptor internalization. Therefore, in neuropathic pain rats, the acetylcholine (ACh)-dependent increase in neuronal excitability is reduced dramatically and the ACh-induced persisting firing, which is critical for working memory, is abolished. We propose that the blunted cholinergic excitability contributes to the functional mPFC deactivation that is causal for the pain phenotype and represents a cellular mechanism for the attention and memory impairments comorbid with chronic pain.

A Rare Variant Identified Within the GluN2B C-Terminus in a Patient with Autism Affects NMDA Receptor Surface Expression and Spine Density.

NMDA receptors (NMDARs) are ionotropic glutamate receptors that are crucial for neuronal development and higher cognitive processes. NMDAR dysfunction is involved in a variety of neurological and psychiatric diseases; however, the mechanistic link between the human pathology and NMDAR dysfunction is poorly understood. Rare missense variants within NMDAR subunits have been identified in numerous patients with mental or neurological disorders. We specifically focused on the GluN2B NMDAR subunit, which is highly expressed in the hippocampus and cortex throughout development. We analyzed several variants located in the GluN2B C terminus and found that three variants in patients with autism (S1415L) or schizophrenia (L1424F and S1452F) (S1413L, L1422F, and S1450F in rodents, respectively) displayed impaired binding to membrane-associated guanylate kinase (MAGUK) proteins. In addition, we observed a deficit in surface expression for GluN2B S1413L. Furthermore, there were fewer dendritic spines in GluN2B S1413L-expressing neurons. Importantly, synaptic NMDAR currents in neurons transfected with GluN2B S1413L in GluN2A/B-deficient mouse brain slices revealed only partial rescue of synaptic current amplitude. Functional properties of GluN2B S1413L in recombinant systems revealed no change in receptor properties, consistent with synaptic defects being the result of reduced trafficking and targeting of GluN2B S1413L to the synapse. Therefore, we find that GluN2B S1413L displays deficits in NMDAR trafficking, synaptic currents, and spine density, raising the possibility that this mutation may contribute to the phenotype in this autism patient. More broadly, our research demonstrates that the targeted study of certain residues in NMDARs based on rare variants identified in patients is a powerful approach to studying receptor function.SIGNIFICANCE STATEMENT We have used a "bedside-to-bench" approach to investigate the functional regulation of NMDA receptors (NMDARs). Using information from deep sequencing of patients with neurological or psychiatric disorders, we investigated missense variants identified in the intracellular C-terminal domain of the GluN2B NMDAR subunit. We found several variants that displayed altered properties. In particular, one variant identified in a patient with autism, human GluN2B S1415L, displayed reduced surface expression and binding to PSD-95. Furthermore expression of GluN2B S1415L (S1413L in mouse) showed a deficit in rescue of synaptic NMDAR currents and fewer dendritic spines, consistent with other reports of spine abnormalities being associated with autism. More broadly, we demonstrate that using patient data is an effective approach to probing the structure/function relationship of NMDARs.

Dynamic Regulation of N-Methyl-d-aspartate (NMDA) and α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptors by Posttranslational Modifications.

Many molecular mechanisms underlie the changes in synaptic glutamate receptor content that are required by neuronal networks to generate cellular correlates of learning and memory. During the last decade, posttranslational modifications have emerged as critical regulators of synaptic transmission and plasticity. Notably, phosphorylation, ubiquitination, and palmitoylation control the stability, trafficking, and synaptic expression of glutamate receptors in the central nervous system. In the current review, we will summarize some of the progress made by the neuroscience community regarding our understanding of phosphorylation, ubiquitination, and palmitoylation of the NMDA and AMPA subtypes of glutamate receptors.

Anti-NMDAR antibodies in new-onset psychosis. Positive results in an HIV-infected patient.

The role of neuronal surface autoantibodies (NSAs) in non-encephalitic psychosis is of recent and controversial interest. Most of the studies relating NSAs with psychosis are retrospective and only focused on the N-methyl-d-aspartate glutamate receptor (NMDAR). Our goal was to evaluate the prevalence of IgG antibodies against the NMDAR NR1 subunit (NMDAR-Abs) along with five additional NSAs in 61 first psychotic episode patients and 47 matched controls. We found two patients positive for NMDAR-Abs (3.3%). One of them was eventually considered to have been misdiagnosed and reclassified as encephalitis. The other met the criteria for bipolar I disorder, presented no neurological symptoms and had a comorbid HIV infection of vertical transmission. This is the first reported case of an HIV-infected patient with psychosis associated with NSAs. This study shows that patients presenting with clinically incomplete forms of anti-NMDAR encephalitis, with predominant or isolated psychiatric symptoms, can remain undetected if no ancillary tests are performed. To improve patient diagnosis and treatment of individuals with a first psychotic episode, more detailed neurological examinations might be needed. Further studies are required to better clarify the role of NSAs in the neuropsychiatric effects of HIV infection.