Enhancing immunotherapy through PD-L1 upregulation: the promising combination of anti-PD-L1 plus mTOR inhibitors.

Immune checkpoint inhibitors (ICIs) targeting the programmed cell death protein 1 (PD-1)/programmed cell death 1 ligand 1 (PD-L1) pathway have transformed urothelial cancer (UC) therapy. The correlation between PD-L1 expression and ICI effectiveness is uncertain, leaving the role of PD-L1 as a predictive marker for ICI efficacy unclear. Among several ways to enhance the efficacy of ICI, trials are exploring combining ICIs with serine/threonine-protein kinase mTOR (mTOR) inhibitors in different tumor types. The potential interaction between mTOR inhibitors and PD-L1 expression in UC has not been well characterized. In our study, we investigated how phosphoinositide 3-kinase (PI3K)/AKT/mTOR pathway inhibitors (TAK-228, everolimus and TAK-117) affect PD-L1 expression and function in preclinical bladder cancer cell models. TAK-228 increased cell surface levels of glycosylated PD-L1 in all but one of the seven cell lines, regardless of baseline levels. TAK-228 promoted the secretion of epidermal growth factor (EGF) and interferon-ß (IFNß), both linked to PD-L1 protein induction. Blocking EGF and IFNß receptors reversed the TAK-228-induced PD-L1 increase. Additionally, TAK-228 enhanced IFN-γ-induced PD-L1 expression and intracellular HLA-I levels in some cells. TAK-228-treated bladder cancer cells exhibited resistance to the cytotoxic effects of peripheral blood mononuclear cells (PBMCs) and cluster of differentiation 8 (CD8)+ T cells. The addition of an anti-PD-L1 antibody diminished this resistance in T24 cells. Increased expression of PD-L1 under TAK-228 exposure was confirmed in patient-derived explants (PDEs) treated ex vivo. These preclinical findings suggest that mTOR inhibition with TAK-228 can increase PD-L1 levels, potentially impacting the specific immune response against UC cells. This highlights the rationale for exploring the combination of mTOR inhibitors with ICIs in patients with advanced UC.

Timing Matters: Ultra-Early and Early versus Late Thromboprophyaxis Following Spontaneous Intracerebral Hemorrhage: A Systematic Review and Meta-Analysis.

Spontaneous intracerebral hemorrhage (ICH) represents a critical and potentially devastating medical event resulting from the rupture of intracerebral vessels. Patients afflicted with ICH face an increased risk of venous thromboembolism (VTE) due to factors such as immobility. However, determining the ideal timing for initiating venous thromboembolism thromboprophylaxis (TP) remains uncertain, as it may carry the potential risk of exacerbating hematoma expansion. Thus, our objective was to ascertain the optimal timing for initiating TP following ICH through a comprehensive systematic review and meta-analysis.This systematic review and meta-analysis were performed following the Cochrane Collaboration and the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) statement guidelines, considering outcomes based on the time of intervention: Ultra early (UEPT) < 24 h, Early (EPT) < 48 h, Late (LPT) > 48 h to perform an analysis on hematoma expansion and mortality.Of 2.777 Hematoma expansion was not more frequent in the 440 patients receiving UEPT/EPT (n = 440) versus 565 receiving LPT (Odds ratio (OR) 0.94 (95% CI; 0.62 to 1.43; I2 = 0%)). Similarly, mortality was not lower in the 293 received UEPT or EPT versus 477 receiving LPT (OR 0.63 (95% CI; 0.39 to 1.0; I2 = 0%).This study, through a systematic review and meta-analysis, conclusively found no difference in intracranial hematoma expansion and/or increased mortality between the use of heparin in the early thromboprophylaxis (< 48 h) group compared to the late thromboprophylaxis (> 48 h) group. Implementing this approach in the management of spontaneous cerebral hemorrhage could facilitate progress towards more optimal care protocols.

Rentabilidad de la ratio angiogénica en el seguimiento de gestantes de alto riesgo de preeclampsia precoz tratadas con ácido acetilsalicílico / Profitability of the angiogenic ratio in the follow-up of pregnant women at high risk of early preeclampsia treated with acetylsalicylic acid

Objetivo: Evaluar el valor predictivo negativo de la ratio antigénica y conocer su rentabilidad para descartar preeclampsia precoz en pacientes de alto riesgo de desarrollarla, con profilaxis de ácido acetilsalicílico. Métodos: Se realizó un estudio descriptivo transversal que recogió a las gestantes con cribado de preeclampsia precoz de alto riesgo (384 gestantes) en el Hospital Santa Lucía durante el año 2021, para lo que se usó test Elecsys® tabulado a un riesgo mayor a 1/150 en primer trimestre, y que tomaran ácido acetilsalicílico antes de la semana 16, quedando en 368 gestantes vistas en las semanas 20, 26, 31 y 36. Se realizó biometría, ratio angiogénica y doppler. Resultados: La incidencia de preeclampsia precoz en la población fue 4 casos (incidencia 1,08 %). Son significativos por su alto valor predictivo negativo del 100 % de preeclampsia precoz: la ratio angiogénica mayor a 38 en la semana 26 y el doppler de las uterinas en semana 20 y 26. Conclusión: En gestaciones con cribado de alto riesgo de preeclampsia que tomen ácido acetilsalicílico, una ratio angiogénica menor a 38 en la semana 26, además de un doppler uterino normal en semana 20 y 26 permite reducir el seguimiento gestacional(AU)

Objective: Our main objective was to evaluate the negative predictive value of the angiogenic ratio and to know its profitability to rule out early preeclampsia in patients at high risk of early preeclampsia with acetylsalicylic acid prophylaxis. Methods: A cross-sectional descriptive study was carried out that included pregnant women with high-risk early preeclampsia screening (384 pregnant women) at the Santa Lucía Hospital during the year 2021, for which the Elecsys® test tabulated at a risk >1/ was used. 150 in the first trimester, and who take acetylsalicylic acid before week 16, leaving 368 pregnant women seen in weeks 20, 26, 31 and 36, with biometry, angiogenic ratio and Doppler performed. Results: The incidence of early preeclampsia in the population was 4 cases (incidence 1.08%). They are significant due to their high negative predictive value of 100% of early preeclampsia: Angiogenic ratio > 38 in week 26, uterine Doppler in weeks 20 and 26. Conclusion: Pregnancies with high risk screening for preeclampsia who take acid acetylsalicylic acid, an angiogenic ratio < 38 at week 26 in addition to a normal uterine Doppler at weeks 20 and 26 allows for reduced gestational follow-up(AU)

Left Internal Capsule Cavernoma Using the Superior Frontal Sulcus as a Surgical Corridor: Why and How to Do It: 2-Dimensional Operative Video.

Cavernous angioma is a sinusoidal dilatation covered by a single layer of endothelium, separated by a collagen matrix with elastin and smooth muscle.1 The prevalence in the general population is estimated at 0.4% to 0.9%,2 representing around 5% to 10% of all vascular malformations.3 Studies indicate 9% to 35% of cavernomas are found in deep locations such as the brainstem, thalamus, and basal ganglia.4-6 Common symptoms of these deep lesions are cranial nerve deficit, hemiparesis, and paresthesia. These lesions have high rates of rebleeding after the first episode of bleeding but present excellent results of surgical resection and modified Rankin in the long term.7-13 Internal capsule cavernomas are particularly challenging due to the important projection fibers surround them. Although the gold standard of treatment is microsurgery, there needs to be a consensus on the best approach for lesions of this topography. We present a video case of a female in her 50s with right hemiparesis and dysphasia, exhibiting grade 3/5 strength on the right side. T1 MRI revealed a high intensity, heterogeneous, multinodular signal in the left basal ganglia, with tractography showing the lesion dividing the posterior limb of the internal capsule amid fibers of the right corticospinal tract. The patient consented to the procedure and to the publication of his/her image. This study was approved by the Ethics and Research Committee of our institution. We demonstrated that the superior frontal sulcus is a safe corridor to surgically cure cavernomas of the internal capsule, with the recovery of previous deficits.

Model of Arteriovenous Malformation Created in Human Placenta for Training in Vascular Microneurosurgery.

BACKGROUND AND OBJECTIVE: Arteriovenous malformations (AVMs) are congenital lesions, and because of their structure, complexity, flow, size, and location organization, they are lesions that require extensive anatomic knowledge and mastery of microsurgical skills and techniques. Human placentas as a training model for AVM surgery are promising alternatives. This article aims to describe the technique for forming an AVM-type lesion in human placentas and its usefulness in the training of microsurgical treatment techniques. METHODS: In this study, 15 fresh human placental models were treated. A nidus was created using synthetic material, and dynamic flow was evaluated with intravascular injection of Indocyanine Green. The catheter system was connected to a continuous flow infusion pump. For simulation purposes, 4 vascular neurosurgeons and 4 vascular neurosurgery fellows used the same techniques and instruments used in real surgery to simulate the resection of AVM lesions. Subjective assessments were conducted, evaluating the validity and structured content on a 5-point Likert scale. Evaluation criteria included the execution of technical maneuvers and the model's expression and structural aspects. RESULTS: We describe the step-by-step creation of an AVM in a placental biological model for the performance of vascular microsurgery training in the laboratory. We created in the human placenta a lesion with the characteristics of an AVM for microsurgical training in the laboratory, which presents key features realistic to a real AVM, such as 1 or more feeder arteries, nidus (synthetic), draining vein(s), continuous and pulsatile flow, and 3-dimensional configuration. Furthermore, it demonstrates the applicability of microsurgical techniques to the model compared with performing surgery on a patient. CONCLUSION: Considering it an effective method for laboratory training, the creation of arteriovenous malformations in human placentas enables students to replicate, comprehend the structure, and master microsurgical techniques in a realistic model.

Genotypic Identification of Trees Using DNA Barcodes and Microbiome Analysis of Rhizosphere Microbial Communities.

DNA barcodes can provide accurate identification of plants. We used previously reported DNA primers targeting the internal transcribed spacer (ITS1) region of the nuclear ribosomal cistron, internal transcribed spacer (ITS2), and chloroplast trnL (UAA) intron to identify four trees at Bergen Community College. Two of the four trees were identified as Acer rubrum and Fagus sylvatica. However, Quercus was only identified at the genus level, and the fourth tree did not show similar identification between barcodes. Next-generation sequencing of 16S rRNA genes showed that the predominant bacterial communities in the rhizosphere mainly consisted of the Pseudomonadota, Actinomycetota, Bacteroidota, and Acidobacteriota. A. rubrum showed the most diverse bacterial community while F. sylvatica was less diverse. The genus Rhodoplanes showed the highest relative bacterial abundance in all trees. Fungal ITS sequence analysis demonstrated that the communities predominantly consisted of the Ascomycota and Basidiomycota. Quercus showed the highest fungi diversity while F. sylvatica showed the lowest. Russula showed the highest abundance of fungi genera. Average similarity values in the rhizosphere for fungi communities at the phylum level were higher than for bacteria. However, at the genus level, bacterial communities showed higher similarities than fungi. Similarity values decreased at lower taxonomical levels for both bacteria and fungi, indicating each tree has selected for specific bacterial and fungal communities. This study confirmed the distinctiveness of the microbial communities in the rhizosphere of each tree and their importance in sustaining and supporting viability and growth but also demonstrating the limitations of DNA barcoding with the primers used in this study to identify genus and species for some of the trees. The optimization of DNA barcoding will require additional DNA sequences to enhance the resolution and identification of trees at the study site.

Sinus Venosus Atrial Septal Defect as an Overlooked Source of Shortness of Breath Among Patients With Pulmonary Arterial Hypertension.

Sinus venosus atrial septal defects (SVASD) associated with partial anomalous pulmonary venous return (PAPVR) can be overlooked as a source of dyspnea in adult patients with pulmonary hypertension. We present the case of a 61-year-old male with exertional dyspnea initially attributed to pulmonary hypertension, who was subsequently diagnosed with SVASD and right superior PAPVR. This case underscores the critical importance of maintaining high clinical awareness and utilizing multimodal imaging techniques in cardiology to accurately diagnose and manage pulmonary hypertension secondary to congenital heart disease. Timely surgical correction can significantly improve morbidity and mortality outcomes.

N-terminal pro-B-type natriuretic peptide and pulmonary echography are predictors of acute heart failure needing early mechanical ventilation in acute coronary syndrome.

AIM: The aim of this study was to determine the best clinical predictors of acute heart failure needing mechanical ventilation (MV) in the first 48 h of evolution of patients admitted because of acute coronary syndrome (ACS). METHODS: We analyzed a cohort of patients admitted for ACS between February 2017 and February 2018. A pulmonary ultrasound was performed on admission and was considered positive (PE+) when there were three or more B-lines in two quadrants or more of each hemithorax. It was compared with N-terminal pro-B-type natriuretic peptide (NT-proBNP), peak troponin T-us value GRACE (Global Registry of Acute Coronary Events), CRUSADE (Can Rapid risk stratification of Unstable angina patients Suppress ADverse outcomes with Early implementation of the American College of Cardiology and American Heart Association guidelines - Bleeding Score), CACS (Canada Acute Coronary Syndrome risk score), and HAMIOT (Heart Failure after Acute Myocardial Infarction with Optimal Treatment score) scores, shock index, ejection fraction, chest X-ray, and Killip class at admission as predictors of MV in the first 48 h of admission. RESULTS: A total of 119 patients were included: 54.6% with ST elevation and 45.4% without ST elevation. Twelve patients (10.1%) required MV in the first 48 h of evolution. The sensitivity of PE+ was 100% (73.5-100%), specificity 91.6% (84.6-96.1%), and area under the curve was 0.96 (0.93-0.96). The sensitivity of an NT-proBNP value more than 3647 was 88.9% (51.9-99.7%), specificity 92.1% (84.5-96.8%), and area under the curve was 0.905 (0.793-1). The κ statistic between both predictors was 0.579. All the other scores were significantly worse than PE + . CONCLUSION: Lung ultrasound and a high NT-proBNP (3647 ng/L in our series) on admission are the best predictors of acute heart failure needing MV in the first 48 h of ACS. The agreement between both tests was only moderate.

Microsurgical Resection of a Left Temporal Stem Cavernous Malformation Through Transsylvian Approach: 2-Dimensional Operative Video.

Cavernous malformations (CMs) are rare, often oligosymptomatic vascular lesions. Common manifestations include seizures and focal neurological deficits. Depending on the symptoms, location, size, and risk factors of bleeding, such as the presence of a developmental venous anomaly, the injury can be highly morbid. Hence, one can consider surgical resection. Deep and eloquently located CMs, such as those located in the temporal trunk, can be quite challenging and require an exact operative technique.1-7 We present a 27-year-old patient with a history of headaches that began two years ago and significantly worsened in the last month, associated with visual blurring, scotomas, nausea, phonophobia, and photophobia as well as paresthesias in the hands and perioral region. Imaging investigation revealed a CM in the temporal stem (Zabramski classification II). Owing to the risk of rebleeding and the young age, surgical resection was performed using a transsylvian approach to preserve the temporal cortex. We describe the technique applied and demonstrate the necessary care manipulating the distal sylvian fissure and the superficial sylvian veins. We also detail the anatomy of the temporal stem and the benefit of the transsylvian approach to preserve the white matter fibers that compose the temporal stem. The patient consented to the procedure and to the publication of his/her image. This study was approved by the Ethics Committee of our institution. Performed CM resection using the transsylvian pterional craniotomy technique, and it proceeded without complications. The postoperative period was also uneventful. The postoperative imaging demonstrated total resection of the cavernoma.

Superior frontal sulcus: a non-eloquent corridor for cavernomas of the internal capsule.

Introduction: Deep cavernomas of eloquent areas, located in the region of the basal nuclei and thalamus, account for 9 to 36% of these encephalic vascular malformations. Internal capsule cavernomas are particularly challenging, as they are surrounded by important projection fibers and their manipulation can lead to permanent deficits. To demonstrate through surgical cases that cavernomas of the internal capsule can be approached by frontal craniotomy, via the superior frontal sulcus, in a curative manner and with low morbidity. Methods: We presented two cases of cavernomas of the internal capsule operated, whose treatment was microsurgical resection via frontal craniotomy and access to the lesion via the superior frontal sulcus, described step-by-step. To elucidate the rationale behind the decision, we used preoperative images with an emphasis on the patients' tractography and the importance of comparing these images with anatomical specimens dissected in the neuroanatomy and microsurgery laboratory. Results: The two cases of internal capsule cavernomas, one in the anterior limb and the other in the posterior limb, were treated surgically via the superior frontal sulcus. Discussion: Both patients showed radiological cure and clinical improvement in the post-operative segment. The patient consented to the procedure and to the publication of his/her image. Treatment of internal capsule cavernomas via the superior frontal sulcus has proven to be a safe and effective option.

Trace gas oxidation sustains energy needs of a thermophilic archaeon at suboptimal temperatures.

Diverse aerobic bacteria use atmospheric hydrogen (H2) and carbon monoxide (CO) as energy sources to support growth and survival. Such trace gas oxidation is recognised as a globally significant process that serves as the main sink in the biogeochemical H2 cycle and sustains microbial biodiversity in oligotrophic ecosystems. However, it is unclear whether archaea can also use atmospheric H2. Here we show that a thermoacidophilic archaeon, Acidianus brierleyi (Thermoproteota), constitutively consumes H2 and CO to sub-atmospheric levels. Oxidation occurs across a wide range of temperatures (10 to 70 °C) and enhances ATP production during starvation-induced persistence under temperate conditions. The genome of A. brierleyi encodes a canonical CO dehydrogenase and four distinct [NiFe]-hydrogenases, which are differentially produced in response to electron donor and acceptor availability. Another archaeon, Metallosphaera sedula, can also oxidize atmospheric H2. Our results suggest that trace gas oxidation is a common trait of Sulfolobales archaea and may play a role in their survival and niche expansion, including during dispersal through temperate environments.

Immunogens in Balamuthia mandrillaris: a proteomic exploration.

Balamuthia mandrillaris is the causative agent of granulomatous amoebic encephalitis, a rare and often fatal infection affecting the central nervous system. The amoeba is isolated from diverse environmental sources and can cause severe infections in both immunocompromised and immunocompetent individuals. Given the limited understanding of B. mandrillaris, our research aimed to explore its protein profile, identifying potential immunogens crucial for early granulomatous amoebic encephalitis diagnosis. Cultures of B. mandrillaris and other amoebas were grown under axenic conditions, and total amoebic extracts were obtained. Proteomic analyses, including two-dimensional electrophoresis and mass spectrometry, were performed. A 50-kDa band showed a robust recognition of antibodies from immunized BALB/c mice; peptides contained in this band were matched with elongation factor-1 alpha, which emerged as a putative key immunogen. Besides, lectin blotting revealed the presence of glycoproteins in B. mandrillaris, and confocal microscopy demonstrated the focal distribution of the 50-kDa band throughout trophozoites. Cumulatively, these observations suggest the participation of the 50-kDa band in adhesion and recognition mechanisms. Thus, these collective findings demonstrate some protein characteristics of B. mandrillaris, opening avenues for understanding its pathogenicity and developing diagnostic and therapeutic strategies.

Posterior Transtemporal Approach to a Thalamic Cavernous Malformation: 2-Dimensional Operative Video.

Cavernous malformations (CMs) account for 10%-15% of all vascular malformations and represent the second most common type of cerebral vascular lesion.1 They typically occur in the cerebral subcortex or white matter.2 CMs located in the thalamus are rare.3 When we isolate the group of thalamic CMs, we find a bleeding risk of >5% per year, with a rebleeding rate exceeding 60%, often occurring within 1 year of the initial bleeding.1 The deep location and proximity to eloquent brain regions make thalamic CMs challenging for neurosurgeons.4,5 Surgeons can access the posterolateral thalamus through various surgical approaches, such as transcallosal transventricular, supracerebellar transtentorial, intraparietal sulcus, and transcortical methods. Selecting the best surgical approach requires considerable expertise, considering the patient's preoperative condition and the lesion's location.6-12 We discuss a complex case involving a 24-year-old patient with a right thalamic cavernoma and a history of 3 prior bleeding events. We present a step-by-step transcortical approach through the posterior portion of the superior temporal gyrus (Video 1). The patient consented to the procedure and publication of images. We demonstrate how the transtemporal posterior trajectory provides an optimal working corridor for safely removing this cavernous malformation without introducing new deficits.

The Technique for Transorbital Ventricular Puncture: An Anatomic Approach.

BACKGROUND AND OBJECTIVES: Transorbital ventricular puncture is a minimally invasive described procedure with poor landmarks and anatomic references. This approach can be easily performed to save patients with intracranial hypertension, especially when it is secondary to an acute decompensated hydrocephalus. This study aims to describe anatomic structures and landmarks to facilitate the execution of transorbital puncture in emergency cases. METHODS: We analyzed 120 head computed tomographies to show the best area to perform the procedure in the orbital roof. Two adult cadavers (4 sides) were punctured in the predetermined area. Angles, distances, landmarks, and anatomic structures were registered. This approach to the ventricular system may be performed at bedside to relieve intracranial hypertension only in specific cases. RESULTS: The perforation point is 2.5 cm (female) or 3.0 cm (male) lateral to the midline and immediately inferior to the superciliary arch. A vertical line, parallel to midline, was drawn on the outer edge of the patient's forehead, the needle was 45° inferiorly and 20° medially and then progressed 2.0 cm backwards to reach the bone perforation point. After that, it was advanced another 4.5cm approximately until it reached the anterior horn of the lateral ventricle. CONCLUSION: Based on statistical and experimental evidences, we were able to establish reliable anatomic reference points to access the anterior horn of the lateral ventricle through transorbital puncture.

Topographical Systematization of Human Placenta Model for Training in Microneurosurgery.

BACKGROUND: Neurosurgical training continuously seeks innovative methods to enhance the acquisition of essential technical skills for neurosurgeons worldwide. While various training models have been employed, few truly replicate real-life conditions optimally. Human placenta is a good model for neurosurgical microsurgery training due to its anatomic similarities to neurovascular structures. Placental vessels exhibit a branching pattern and caliber comparable with intracranial vessels, making them suitable for practicing microsurgical techniques. The study aims to delineate the anatomic zones of the placenta and propose a segmented training model, resulting in a reproducible, cost-effective, and realistic neurosurgical microsurgery training environment. METHODS: Twenty human placentas were meticulously prepared, injected with dyes, and categorized into zones on the basis of anatomic features. Measurements of placental vessels were recorded and compared with cerebral vessels. The placenta was divided into 4 quadrants to facilitate specific training techniques. RESULTS: Our results revealed varying vessel diameters across placental zones, closely resembling cerebral vessels. Different microsurgical techniques were applied to specific placental zones, thereby optimizing training scenarios. The applicability section described exercises such as membrane dissection, vessel skeletonization, aneurysm creation, vascular bypass, and tumor dissection within the placental model, providing detailed guidance on the zones suitable for each exercise. CONCLUSIONS: Human placenta serves as an effective microsurgical training model for neurosurgery, enhancing neurosurgeons' skills through anatomic segmentation. Integrating this model into training programs can significantly contribute to skill acquisition and improved surgical outcomes. Further research is warranted to refine and expand its utilization, complemented by clinical experiences and other simulation tools.

Detection and Characterization of Electrogenic Bacteria from Soils.

Soil microbial fuel cells (SMFCs) are bioelectrical devices powered by the oxidation of organic and inorganic compounds due to microbial activity. Seven soils were randomly selected from Bergen Community College or areas nearby, located in the state of New Jersey, USA, were used to screen for the presence of electrogenic bacteria. SMFCs were incubated at 35-37 °C. Electricity generation and electrogenic bacteria were determined using an application developed for cellular phones. Of the seven samples, five generated electricity and enriched electrogenic bacteria. Average electrical output for the seven SMFCs was 155 microwatts with the start-up time ranging from 1 to 11 days. The highest output and electrogenic bacterial numbers were found with SMFC-B1 with 143 microwatts and 2.99 × 109 electrogenic bacteria after 15 days. Optimal electrical output and electrogenic bacterial numbers ranged from 1 to 21 days. Microbial DNA was extracted from the top and bottom of the anode of SMFC-B1 using the ZR Soil Microbe DNA MiniPrep Protocol followed by PCR amplification of 16S rRNA V3-V4 region. Next-generation sequencing of 16S rRNA genes generated an average of 58 k sequences. BLAST analysis of the anode bacterial community in SMFC-B1 demonstrated that the predominant bacterial phylum was Bacillota of the class Clostridia (50%). However, bacteria belonging to the phylum Pseudomonadota (15%) such as Magnetospirillum sp. and Methylocaldum gracile were also part of the predominant electrogenic bacterial community in the anode. Unidentified uncultured bacteria accounted for 35% of the predominant bacterial community. Bioelectrical devices such as MFCs provide sustainable and clean alternatives to future applications for electricity generation, waste treatment, and biosensors.