Evaluation of the Vascular Endothelial Growth Factor and Smooth Muscle Actin Expression With Microvessel Density and Morphometric Analysis of Endometrial Vessels in Patients With Abnormal Uterine Bleeding.

BACKGROUND: Abnormal uterine bleeding (AUB) that occurs in a structurally normal uterus with regular menstrual cycles and without other identifiable etiology is often caused by a primary endometrial disorder (AUB-E). Altered vascular morphological changes and expression of markers of angiogenesis have been implicated as an underlying cause in these cases. OBJECTIVES:  The study was conducted to investigate the expression of vascular endothelial growth factor (VEGF) and smooth muscle actin-alpha (SMA-α), and to perform microvessel density (MVD), and morphometric evaluation of endometrial vessels in patients with AUB-E. MATERIAL AND METHODS: Endometrial biopsies and hysterectomy specimens of 40 patients clinically diagnosed with AUB-E were included in the study with 40 age-matched controls. Immunohistochemistry (IHC) with VEGF and SMA-α was performed, and the expression and staining pattern was recorded as the number of positive vessels per 10 high power fields and intensity scores. Morphometric analysis was performed on CD34 stained sections using Leica Application Suite, version 4.4.0 software (Leica Microsystems, Wetzlar, Germany). MVD was calculated by the vascular hotspot method. RESULTS: A statistically significant increase in VEGF vessel count (p-value<0.001) and a decline in SMA-α expression (p-value=0.23) was seen in cases as compared to the control group. There was a statistically significant increase in microvessel caliber (p-value=0.01) and MVD (p-value <0.001) in cases as compared to controls. CONCLUSION: These findings support aberrant vascular proliferation and impaired vessel maturation, contributing to the pathology of AUB-E. Alterations in angiogenesis in these patients reveal potential therapeutic targets for AUB.

The spectrum of microvascular patterns in adult diffuse glioma and their correlation with tumor grade.

BACKGROUND: Primary brain tumors constitute the leading cause of cancer-related mortality. Among them, adult diffuse gliomas are the most common type, affecting the cerebral hemispheres and displaying a diffuse infiltrative pattern of growth in the surrounding neuropil that accounts for about 80% of all primary intracranial tumors. The hallmark feature of gliomas is blood vessel proliferation, which plays an important role in tumor growth, tumor biological behavior, and disease outcome. High-grade gliomas exhibit increased vascularity, the worst prognosis, and lower survival rates. Several angiogenic receptors and factors are upregulated in glioblastomas and stimulate angiogenesis signaling pathways by means of activating oncogenes and/or down-regulating tumor-suppressor genes. Existing literature has emphasized that different microvascular patterns (MVPs) are displayed in different subtypes of adult diffuse gliomas. METHODS: We examined the distribution and biological characteristics of different MVPs in 50 patients with adult diffuse gliomas. Haematoxylin and eosin staining results, along with periodic acid-Schiff and CD34 dual-stained sections, were examined to assess the vascular patterns and correlate with different grades of diffuse glioma. RESULTS: The present observational study on adult diffuse glioma evaluated tumor grade and MVPs. Microvascular sprouting was the most common pattern, while a bizarre pattern (type 2) was associated with the presence of a high-grade glioma. Vascular mimicry was observed in 6% of cases, all of which were grade 4 gliomas. CONCLUSIONS: This study supplements the role of neo-angiogenesis and aberrant vasculature patterns in the grading and progression of adult diffuse gliomas, which can be future targets for planning treatment strategies.

Diagnostic Utility of Direct Immunofluorescence on Paraffin-Embedded Skin Biopsy Samples for the Diagnosis of Autoimmune Vesiculobullous Lesions.

Background Autoimmune vesiculobullous diseases (AIBDs) are a group of diseases characterized by blisters of the skin/mucosa due to the presence of circulating autoantibodies against antigens in the epidermis or the dermo-epidermal junction. Direct immunofluorescence (DIF) for immunoglobulin (Ig)G, IgC3, and IgA on fresh-frozen tissue is the gold standard diagnostic test for AIBDs. However, DIF in the absence of frozen tissue is challenging for the diagnosis of AIBDs. This study aimed to analyze the practical utility of DIF using paraffin-embedded skin biopsy rather than fresh frozen tissue for the diagnosis of AIBDs. Methodology This cross-sectional comparative study included 30 cases of AIBDs. DIF for IgG and IgA was performed on paraffin-embedded tissue (PE-DIF) after proteinase digestion on histopathologically confirmed 15 pemphigus vulgaris (PV), three pemphigus foliaceous (PF), four bullous pemphigoid (BP), three dermatitis herpetiformis (DH), three subcorneal pustular dermatosis (SCPD), and one case each of linear IgA disease and pemphigoid gestationis (PG). PE-DIF staining pattern was compared with the DIF on fresh frozen tissue (FF-DIF). Results All cases of PV and PF showed an intercellular IgG chicken wire staining pattern similar to FF-DIF. However, background staining was more intense in PV cases while less intense in PF cases. Three BP cases showed linear IgG staining in PE-DIF. DH, SCPD, linear IgA disease, and PG cases did not show IgG positivity. Out of three DH cases, two cases showed granular IgA positivity while linear IgA positivity along the basement membrane was seen in a single case of linear IgA disease. Negative IgG staining was observed in SCPD. Immunofluorescence in PE-DIF was rapidly deteriorating than in FF-DIF. Conclusions DIF done on paraffin-embedded tissue can be used as a supplement and salvage technique with histopathology for the diagnosis of AIBDs, particularly when a cryostat facility for frozen tissue is not available and the patient is unable to undergo a second biopsy procedure.

Development of a cost-effective compact diode-laser-based photoacoustic sensing instrument for breast tissue diagnosis.

Significance: The photoacoustic (PA) technique, a noninvasive pump-probe technique, has found interesting applications in biomedical tissue diagnosis over the last decade. To take it a step further to clinical applications, the PA technique needs to be designed as an instrument focusing on a compact design, reducing the cost, and quickly providing a quantitative diagnosis. Aim: This work presents a design and characterization of a cost-effective, compact PA sensing instrument for biomedical tissue diagnosis. Approach: A compact laser diode case design is developed to house several laser diodes for PA excitation, and a pulsed current supply unit is also developed in-house to power the laser diodes to generate a 25 ns current pulse at a frequency of 20 kHz. After PA experimental data acquisition, the signal's frequency spectra were calculated to characterize the tissue quantitatively and correlated with their mechanobiological properties. Results: The corresponding dominant frequency peak in the PA spectral response (PASR) study was low in the fibrofatty normal breast tissue 0.26±0.03 MHz, compared to the dominant frequency peak of 1.60±0.016 MHz in the fibrocystic disease tissue, which had increased glandular and stromal elements, thereby increased tissue density. The histopathological findings correlated with the PASR results, and the fibrocystic breast disease tissue exhibited a higher dominant frequency peak and energy compared to the normal breast tissue. Conclusions: We experimented with an in vitro PASR study of fibrocystic human breast tissues and successfully differentiated different tissue types using quantitative spectral parameters peak frequency, mean frequency, and spectral energy. This gives the potential to take this technique further for cost-effective and quick clinical applications.

Cytohistological findings and diagnostic challenges in rare pediatric pulmonary mesenchymal malignancies: A report of two cases.

Pulmonary mesenchymal tumors are uncommon neoplasms and the data available on their clinical, cytohistomorphological, immunohistochemical, and molecular findings are limited, leading to difficulty in timely diagnosis and management. Case 1: A 12-year-old boy presented with a right endobronchial mass. Imprint smears from endobronchial biopsy revealed moderately pleomorphic spindle cell tumor arranged in fascicles and perivascular pattern with attached myxoid material showing occasional mitotic activity suggesting a cytological diagnosis of sarcoma. Biopsy also displayed similar morphology. Extensive immunohistochemistry (IHC) showed diffuse SMA, focal AE1/AE3, and diffuse ALK positivity along with a MIB/Ki67 index of 30%-40% leading to the diagnosis of inflammatory myofibroblastic tumor (IMT; Sarcoma grade). Case 2: A 8-year-old boy presented with a huge left-sided mass replacing the entire lung parenchyma and eroding adjoining ribs. Pleural fluid cytology revealed vague clusters of PAS-positive diastase-sensitive small atypical cells with associated inflammatory cells in the hemorrhagic background, suggesting a diagnosis of malignant small round blue cell tumor. Trucut biopsy from the mass showed spindled to round cells showing diffuse positivity with CD99 and BCL-2. Molecular studies with reverse transcription-PCR (RT-PCR) for SYT-SSX and EWS-FLI1 were negative for synovial sarcoma and Ewing's sarcoma, respectively. Given the clinical setting, PAS positivity, IHC, and molecular studies, the diagnosis of tumors of uncertain differentiation with the possibility of Ewing's sarcoma family of tumors (ESFT) with a translocation between EWS1 and other ETS-family members (ERG, FEV, ETV1, E1AF, etc.) was suggested.

Atypical Morphology of a Typical Mycobacterial Infection.

A-24-year-old woman reported with asymptomatic facial lesions present for 6 months. Examination revealed two closely located nodules which were firm, nontender, slightly erythematosus with crusting over the left cheek (Figure 1A). There was no regional lymphadenopathy, and the systemic examination was within normal limits. The differential diagnosis included cutaneous leishmaniasis, keratoacanthoma, and basal cell carcinoma. Tissue smear from nodules failed to reveal Leishmania donovan bodies. The histopathologic examination revealed nonca-seating epithelioid granulomas with lymphocyte cuffing in the dermis (Figures 2A and 2B). Special staining performed with Ziehl-Neelsen and Periodic acid-Schiff (PAS) stains was negative. Tissue cultures for bacteria, mycobacteria, and fungi were also negative; however Mantoux test (MT) performed for latent tuberculosis was strongly positive. Sputum for acid fast bacilli was negative, and serology for human immuno-deficiency virus (HIV)-1 and HIV-2 was nonreactive. A chest x-ray and ultrasound of the abdomen did not reveal any abnormality. Although the morphology of skin lesions did not favor classic lupus vulgaris (LV), considering the endemicity of tuberculosis in India, positive results of Mantoux test, and a dermal epithelioid granuloma, the patient was prescribed antitubercular therapy (ATT), comprising isoniazid, rifampicin, ethambutol, and pyrazinamide. Dramatic response was observed after 2 months, and complete healing with residual scarring took place in next 4 months (Figure 1B).

Primary Spinal Intradural Extramedullary Ewing's Sarcoma/Peripheral Neuroectodermal Tumour Masquerading Clinically as a Neurogenic Tumour: A Case Report and Review of Literature.

Ewing's Sarcomas (ES)/Peripheral neuroectodermal tumour (pPNET) are heterogenous group of rare, highly malignant, undifferentiated primitive round-cell neoplasms of neuroectodermal origin. pPNETs are seldom observed to involve the spine of which Spinal Intradural Extramedullary Extraosseous Primary ES/pPNET are extremely rare. We report a case of a 23-year-old male with complaints of low backache and hip pain radiating to the left inguinal region for four months. Radiology findings were suggestive of a neurogenic tumour. Cytomorphology, histomorphology and immunohistochemistry evaluation were done. Diagnosis was consistent with ES/pPNET. Careful correlation between clinical history, cytomorphology, histopathology, immunohistochemical and molecular analysis can help to distinguish primary spinal ES/PNET from other primary spinal tumours and will help clinicians to start treatment at the earliest.

Unraveling the Enigma of Perforating Itches: A Comprehensive Report of Reactive Perforating Collagenosis in Three Patients.

Reactive perforating collagenosis (RPC) is a rare dermatosis where dermal connective tissue erupts through the epidermis, resulting in diverse clinical manifestations such as umbilicated papules with crusting and excoriated nodules with central puncta. Associated with systemic disorders like diabetes mellitus, chronic kidney diseases, and autoimmune conditions, RPC's pathogenesis involves abnormal collagen metabolism, immune dysfunction, genetic predisposition, and environmental triggers. Histopathological examination reveals vertically oriented shallow cup-shaped invaginations containing degenerated collagen fibers, aiding diagnosis. Treatment includes managing underlying causes and utilizing options like topical corticoids, retinoids, and phototherapy, with a possibility of spontaneous regression and recurrence. This case report highlights the significance of considering RPC in patients with characteristic skin lesions and severe itching, emphasizing early recognition and accurate diagnosis to optimize patient care. Continued research and collaboration are crucial for improving outcomes in individuals affected by RPC.

Atypical Hansen's Disease Mimicking Xanthogranuloma: Role of Cytology.

Fine needle aspiration cytology is a simple, safe, and effective technique that can be used in the diagnosis of cutaneous diseases. Here, we present a case of Hansen's disease with an erythematous dermal nodule mimicking xanthogranuloma clinically. In our scenario, since leprosy is considered eliminated in India, the presentation of patients with classical signs and symptoms is becoming rare. Atypical manifestation of leprosy is increasing day by day, so it is necessary to have a high suspicion of leprosy in every case.

Bronchoalveolar Lavage in Diagnostic Evaluation of Pulmonary Diseases- An Institutional Experience.

Background: Bronchoalveolar lavage (BAL) is a widely accepted investigative tool for the diagnosis of pulmonary lesions. This study was done to find out the usefulness of BAL in diagnosis of pulmonary lesions from central Indian patient population. Methods: A cross-sectional prospective study was performed over a period of three years. All the BAL specimens of patients presenting to Department of Pulmonary Medicine and Tuberculosis during a period of January 2017 to December 2019 were included in the study. Cyto-histopathologic correlation was done, wherever available. Results: Of total 277 cases, there were 178 (64.5%) males and 99 (35.5%) females. The age of patients ranged from 4 years to 82 years. In 92 (33%) cases, specific infective etiology could be identified on BAL cytology, the most common being tuberculosis (26%) followed by fungal infections (2%). Rarely, infections like nocardia, actinomycosis, and hydatidosis were also identified. Eight cases (3%) of malignancy were identified which included two cases of adenocarcinoma, one case of small cell carcinoma, three cases of poorly differentiated carcinoma, and two cases suspicious for malignancy. Some rare diagnoses like diffuse alveolar damage, pulmonary alveolar microlithiasis, and pulmonary alveolar proteinosis could be identified on BAL. Conclusion: BAL is useful in primary diagnosis of infections and malignancies of lower respiratory tract. BAL may aid in diagnostic workup of diffuse lung diseases. A combination of clinical information, high-resolution computed tomography, and BAL analysis may furnish an assured diagnosis to the clinician and obviate need for invasive procedures.

Conjunctival "Melanoma-In-Situ''-A Rare Neoplasia of Xeroderma Pigmentosum in Indian Scenario.

A 16-year-old girl born from a nonconsanguineous marriage presented to the dermatology out-patient department with innumerable hyperpigmented macules predominantly over the photoexposed parts of her body involving the face, neck, chest, back, of forearms, hands, and legs with a history of photosensitivity and photophobia (Figure 1). These were interspersed with multiple depigmented macules over her arms and legs since, she was three years old. She also had a small pigmented mass involving the left eye present for the last 3 months. She had a family history of similar cutaneous lesions in her elder sister. There was no history of hearing loss, seizures, spasticity, or cognitive impairment.

Evaluation of the Cytonucleomorphometric Parameters for Cases Diagnosed as Squamous Cell Abnormality on Conventional Cervico-Vaginal Pap Smears.

Background and Aim: The natural history of cervical cancer is unique that it is preceded by a precancerous condition for a long time. Morphometry as a tool can be used in early and accurate diagnosis of these precancerous and cancerous lesions. The present study aims at assessing the utility of cellular and nuclear morphometry in differentiating squamous cell abnormality from benign conditions and also differentiating the categories of squamous cell abnormalities. Materials and Methods: Forty-eight diagnosed cases of squamous epithelial cell abnormality, that is, 10 cases each of atypical squamous cell of undetermined significance (ASC-US), low-grade squamous intraepithelial lesion (LSIL), high-grade SIL (HSIL), and squamous cell carcinoma (SCC) and eight cases of ASC-H (ASC cannot exclude HSIL), were made the sample population and compared with a control population of 10 cases of negative for intraepithelial lesion or malignancy (NILM). Parameters like nuclear area (NA), nuclear perimeter (NP), nuclear diameter (ND), nuclear compactness (NC), cellular area (CA), cellular diameter (CD), cellular perimeter (CP), and nucleocytoplasmic (N/C) ratio were used. Results: There was a significant difference in the six groups of squamous cell abnormality based on NA, NP, ND, CA, CP, and CD (P < 0.05) using one-way analysis of variance. Nuclear morphometry parameters like NA, NP, and ND were found to be the maximum for HSIL, followed by LSIL, ASC-H, ASC-US, SCC, and NILM groups in decreasing order. The mean CA, CP, and CD were found to be the maximum for NILM, followed by LSIL, ASC-US, HSIL, ASC-H, and SCC in decreasing order. On post hoc analysis, the lesions can be divided into three groups: NILM/normal; ASC-US and LSIL; and ASC-H, HSIL, and SCC, based on N/C ratio. Conclusion: In cervical lesions, holistic parameter of cytonucleomorphometry should be taken rather than taking nuclear morphometry only. N/C ratio is a highly statistically significant parameter that can differentiate between low-grade lesions and high-grade lesions.

Multifunctional Ultrasmall Theranostic Nanohybrids Developed by Ultrasonic Atomizer for Drug Delivery and Magnetic Resonance Imaging.

Theranostic nanoparticulate systems (TNPs) have shown potential in addressing problems related to spatial localization and temporally controlled release of drugs with the capabilities of real-time imaging to evaluate the progress of therapy. The current study reports the ultrasonic atomization-led synthesis of in vitro and in vivo evaluations of ultrasmall chitosan-based theranostic nanohybrid formulations with encapsulated doxorubicin (DOX) and iron-oxide magnetic nanoparticles. The nanohybrid particles are characterized using transmission electron microscopy, powder X-ray diffraction, FTIR, DOX encapsulation efficiency, in vitro release, cellular uptake, and toxicity. These formulations were also tested for the capability of invivo tumor reduction and simultaneous magnetic resonance imaging using Swiss albino mice. Ultrasonic atomizer-led synthesis resulted in chitosan-magnetic nanohybrids (CMNPs) having sizes of 15 ± 3 nm which comprise MNP of 10 ± 3 nm. The encapsulation of DOX in CMNP was around 25%, resulting in an 80% sustained release over 10 days at pH 5 and 7. CMNP was also found to be an efficient DOX delivery vehicle tested on cancer cells (HeLa). The CMNPs were able to reduce the tumor volume by 60% in 15 days. The inherent magnetic property and nanoscale size of CMNPs also provided for enhanced contrast efficiency in magnetic resonance imaging of tumors. Thus, such multifunctional theranostic nanoparticles can be an efficient tool for targeted diagnostic and therapeutic success.

Bland Urine Sediment in a Child with Acute Kidney Injury.

Tubulointerstitial nephritis (TIN) or acute interstitial nephritis (AIN) is a renal lesion characterized by inflammatory infiltrate limited to the renal interstitium and tubules. Three-fourths of the cases are drug induced, other causes being systemic and autoimmune diseases, and infections. Various drugs have been implicated, the most common being antibiotics such as ß-lactams. Cephalosporins causing AIN have been reported uncommonly, particularly in children. Although renal biopsy confirms the diagnosis, urinalysis provides pertinent diagnostic clues against the backdrop of the clinico-laboratory profile. The presence of white blood cells, white cell casts, and red blood cells in urine sediment have been described in literature. However, a relatively normal urinalysis may be present in some cases and may pose a diagnostic challenge. We present a case of ceftriaxone-induced AIN in a child with bland urine sediment at initial presentation. To the best of our knowledge, this is the first report of ceftriaxone-induced AIN in the pediatric age group.

Giant cell tumor of soft tissue: A case report.

INTRODUCTION AND IMPORTANCE: Giant cell tumor of soft tissue (GCT-ST) is a rare primary neoplasm of soft tissues. It usually involves superficial and deeper soft tissues of upper and lower extremities, followed by trunk. CASE PRESENTATION: A 28-year-old female, presented with a painful mass in left abdominal wall for three months. On examination, it measured 4 × 4 cm with ill-defined margins. CECT showed ill-defined enhancing lesion deep to muscle planes with possible invasion of peritoneal layer. Histopathology showed multinodular architecture with intervening fibrous septa and metaplastic bony tissue encasing the tumor. Tumor composed of round to oval mononuclear cells and osteoclast like multinucleated giant cells. Mitotic figures were eight per hpf. A diagnosis GCT-ST of anterior abdominal wall was made. Patient was treated with surgery followed by adjuvant radiotherapy. Patient is disease free at one year follow up. CLINICAL DISCUSSION: These tumors mostly involve extremities and trunk and usually presents as a painless mass. Clinical features depend upon the exact location of the tumor. Common differential diagnosis includes tenosynovial giant cell tumors and malignant giant cell tumors of soft tissue and GCT of Bone. CONCLUSION: Diagnosis of GCT-ST is difficult on cytopathology and radiology alone. Histopathological diagnosis should be done to rule out the malignant lesions. Complete surgical resection with clear resection margins is the mainstay of treatment. Adjuvant radiotherapy should be considered in case of incomplete resection. Long follow-up is necessary for these tumors as local recurrence and risk of metastasis cannot be predicted.

Testing the Acceptability and Usability of an AI-Enabled COVID-19 Diagnostic Tool Among Diverse Adult Populations in the United States.

BACKGROUND AND OBJECTIVES: Although at-home coronavirus disease-2019 (COVID-19) testing offers several benefits in a relatively cost-effective and less risky manner, evidence suggests that at-home COVID-19 test kits have a high rate of false negatives. One way to improve the accuracy and acceptance of COVID-19 screening is to combine existing at-home physical test kits with an easily accessible, electronic, self-diagnostic tool. The objective of the current study was to test the acceptability and usability of an artificial intelligence (AI)-enabled COVID-19 testing tool that combines a web-based symptom diagnostic screening survey and a physical at-home test kit to test differences across adults from varying races, ages, genders, educational, and income levels in the United States. METHODS: A total of 822 people from Richmond, Virginia, were included in the study. Data were collected from employees and patients of Virginia Commonwealth University Health Center as well as the surrounding community in June through October 2021. Data were weighted to reflect the demographic distribution of patients in United States. Descriptive statistics and repeated independent t tests were run to evaluate the differences in the acceptability and usability of an AI-enabled COVID-19 testing tool. RESULTS: Across all participants, there was a reasonable degree of acceptability and usability of the AI-enabled COVID-19 testing tool that included a physical test kit and symptom screening website. The AI-enabled COVID-19 testing tool demonstrated overall good acceptability and usability across race, age, gender, and educational background. Notably, participants preferred both components of the AI-enabled COVID-19 testing tool to the in-clinic testing. CONCLUSION: Overall, these findings suggest that our AI-enabled COVID-19 testing approach has great potential to improve the quality of remote COVID testing at low cost and high accessibility for diverse demographic populations in the United States.

Foldscope: Diagnostic Accuracy and Feasibility of its Use in National Malaria Control Program.

Background: Malaria has been an important public health all over the globe. Although conventional light microscopy is the gold standard of diagnosis, light microscopes are heavy, fragile, costly, and electricity dependent. Rapid diagnostic tests (RDTs) have become more popular but perform badly in temperate climate. This is because the RDT kits require maintenance of cold chain for its optimal use. In this regard, there is a recent interest in handheld malaria microscopy at the point of care in the field setting. Foldscopes are cheap, handy, nonfragile, and use mobile camera for illumination. The purpose of the study was to find whether foldscope can be used in the national vector borne disease control program (NVBDCP) in India. Methods: Ten laboratory technicians were trained in identifying malaria parasites using foldscope and their mobiles. Later, they were provided with unassembled foldscope to document their test results for the preidentified malaria slides. The blood smears were stained as per the protocol of NVBDCP. The report of the index test (foldscope microscopy) was compared with the reference test (conventional microscopy). Results: The sensitivity and specificity of the index test was found to be 13.3% (6.257-26.18), specificity of 97.78% (88.43-99.61), positive predictive value 85.71% (48.69-97.43), and negative predictive value 53.01% (42.38-63.38). The devise failure rate and test failure rate were 20% and 11.7%. The kappa agreement between the index and reference microscopy was only 11% and the McNemar P < 0.01. Conclusion: The ×400 foldscope at its present magnification and illumination cannot be utilized in the field under NVBDCP.

Pathological changes in COVID-19 pneumonia on limited post-mortem sampling of lung: A saga of inflammation and thrombosis.

We describe three postmortem open lung-biopsy findings among patients with COVID-19 pneumonia who were on anticoagulant therapy. The spectrum of histopathological findings included lung inflammation in the form of diffuse alveolar damage (DAD) in exudative and organizing phases, with or without pulmonary artery thrombosis in different stages of evolution. This spectrum of inflammation and thrombosis may be indicative of a natural history of severe COVID-19 pneumonia or demonstrative of variation in therapeutics.