Targets Of Interest Research Articles

One-Click-Based Perception for Interactive Image Segmentation.

Existing deep learning-based interactive image segmentation methods have significantly reduced the user's interaction burden with simple click interactions. However, they still require excessive numbers of clicks to continuously correct the segmentation for satisfactory results. This article explores how to harvest accurate segmentation of interested targets while minimizing the user interaction cost. To achieve the above goal, we propose a one-click-based interactive segmentation approach in this work. For this particularly challenging problem in the interactive segmentation task, we build a top-down framework dividing the original problem into a one-click-based coarse localization followed by a fine segmentation. A two-stage interactive object localization network is first designed, which aims to completely enclose the target of interest based on the supervision of object integrity (OI). Click centrality (CC) is also utilized to overcome the overlapping problem between objects. This coarse localization helps to reduce the search space and increase the focus of the click at a higher resolution. A principled multilayer segmentation network is then designed by a progressive layer-by-layer structure, which aims to accurately perceive the target with extremely limited prior guidance. A diffusion module is also designed to enhance the information flow between layers. Besides, the proposed model can be naturally extended to multiobject segmentation task. Our method achieves the state-of-the-art performance under one-click interaction on several benchmarks.

Naturally Occurring Cholinesterase Inhibitors from Plants, Fungi, Algae, and Animals: A Review of the Most Effective Inhibitors Reported in 2012-2022.

Since the development of the "cholinergic hypothesis" as an important therapeutic approach in the treatment of Alzheimer's disease (AD), the scientific community has made a remarkable effort to discover new and effective molecules with the ability to inhibit the enzyme acetylcholinesterase (AChE). The natural function of this enzyme is to catalyze the hydrolysis of the neurotransmitter acetylcholine in the brain. Thus, its inhibition increases the levels of this neurochemical and improves the cholinergic functions in patients with AD alleviating the symptoms of this neurological disorder. In recent years, attention has also been focused on the role of another enzyme, butyrylcholinesterase (BChE), mainly in the advanced stages of AD, transforming this enzyme into another target of interest in the search for new anticholinesterase agents. Over the past decades, Nature has proven to be a rich source of bioactive compounds relevant to the discovery of new molecules with potential applications in AD therapy. Bioprospecting of new cholinesterase inhibitors among natural products has led to the discovery of an important number of new AChE and BChE inhibitors that became potential lead compounds for the development of anti-AD drugs. This review summarizes a total of 260 active compounds from 142 studies which correspond to the most relevant (IC50 ≤ 15 μM) research work published during 2012-2022 on plant-derived anticholinesterase compounds, as well as several potent inhibitors obtained from other sources like fungi, algae, and animals.

Learning Single Spectral Abundance for Hyperspectral Subpixel Target Detection.

Due to the limitation of target size and spatial resolution, targets of interest in hyperspectral images (HSIs) often appear as subpixel targets, which makes hyperspectral target detection still faces an important bottleneck, that is, subpixel target detection. In this article, we propose a new detector by learning single spectral abundance for hyperspectral subpixel target detection (denoted as LSSA). Different from most existing hyperspectral detectors that are designed based on a match of the spectrum assisted by spatial information or focusing on the background, the proposed LSSA addresses the problem of detecting subpixel targets by learning a spectral abundance of the target of interest directly. In LSSA, the abundance of the prior target spectrum is updated and learned, while the prior target spectrum is fixed in a nonnegative matrix factorization (NMF) model. It turns out that such a way is quite effective to learn the abundance of subpixel targets and contributes to detecting subpixel targets in hyperspectral imagery (HSI). Numerous experiments are conducted on one simulated dataset and five real datasets, and the results indicate that the LSSA yields superior performance in hyperspectral subpixel target detection and outperforms its counterparts.

LSOTB-TIR: A Large-Scale High-Diversity Thermal Infrared Single Object Tracking Benchmark.

Unlike visual object tracking, thermal infrared (TIR) object tracking methods can track the target of interest in poor visibility such as rain, snow, and fog, or even in total darkness. This feature brings a wide range of application prospects for TIR object-tracking methods. However, this field lacks a unified and large-scale training and evaluation benchmark, which has severely hindered its development. To this end, we present a large-scale and high-diversity unified TIR single object tracking benchmark, called LSOTB-TIR, which consists of a tracking evaluation dataset and a general training dataset with a total of 1416 TIR sequences and more than 643 K frames. We annotate the bounding box of objects in every frame of all sequences and generate over 770 K bounding boxes in total. To the best of our knowledge, LSOTB-TIR is the largest and most diverse TIR object tracking benchmark to date. We spilt the evaluation dataset into a short-term tracking subset and a long-term tracking subset to evaluate trackers using different paradigms. What's more, to evaluate a tracker on different attributes, we also define four scenario attributes and 12 challenge attributes in the short-term tracking evaluation subset. By releasing LSOTB-TIR, we encourage the community to develop deep learning-based TIR trackers and evaluate them fairly and comprehensively. We evaluate and analyze 40 trackers on LSOTB-TIR to provide a series of baselines and give some insights and future research directions in TIR object tracking. Furthermore, we retrain several representative deep trackers on LSOTB-TIR, and their results demonstrate that the proposed training dataset significantly improves the performance of deep TIR trackers. Codes and dataset are available at https://github.com/QiaoLiuHit/LSOTB-TIR.

Molecular Targets and Therapies for Ampullary Cancer.

Ampullary carcinomas are rare but increasing in incidence. Ampullary cancers have molecular alterations that guide choice of therapy, particularly in nonresectable cases. These alterations can be more common by subtype (intestinal, pancreaticobiliary, or mixed), and next-generation sequencing is recommended for all patients who cannot undergo surgery. In this article, we review the approach to tissue acquisition and consideration for molecular testing. Common molecular targets of interest in ampullary cancer are also discussed in this review, including HER2/ERBB2, HER3, tumor mutational burden, microsatellite instability, KRAS, and germline BRCA and ATM mutations, along with emerging and rarer alterations.

Challenges for the Discovery of Non-Covalent WRN Helicase Inhibitors.

The Werner Syndrome RecQ helicase (WRN) is a synthetic lethal target of interest for the treatment of cancers with microsatellite instability (MSI). Different hit finding approaches were initially tested. The identification of WRN inhibitors proved challenging due to a high propensity for artefacts via protein interference, i. e., hits inhibiting WRN enzymatic activities through multiple, unspecific mechanisms. Previously published WRN Helicase inhibitors (ML216, NSC19630 or NSC617145) were characterized in an extensive set of biochemical and biophysical assays and could be ruled out as specific WRN helicase probes. More innovative screening strategies need to be developed for successful drug discovery of non-covalent WRN helicase inhibitors.

Molecular docking, characterization, ADME/toxicity prediction, and anti-ulcer activity of new quercetin derivatives on indomethacin-induced gastric ulcer in mice

Gastric ulcer (GU) is a serious upper gastrointestinal tract disorder that affects people worldwide. The drugs now available for GU treatment have a high rate of relapses and drug interactions, as well as mild to severe side effects. As a result, new natural therapeutic medications for treating GU with fewer negative side effects are desperately needed. Because of quercetin's (QCT) diverse pharmacological effects and unique structural features, we decided to semi-synthesize new QCT derivatives and test them for antiulcer activity. Docking assays were performed on the synthesized compounds to determine their affinity for TLR-4/MD-2, MyD88/TIR, and NF-κB domains, an important inflammatory pathway involved in GU development and progression. Mice were given oral famotidine (40 mg/kg/day), QCT, QCT pentamethyl (QPM), or QCT pentaacetyl (QPA) (50 mg/kg/day) for 5 days before GU induction by a single intraperitoneal injection of indomethacin (INDO; 18 mg/kg). QPM and QPA have a stronger binding affinity for TLR-4/MD-2, MyD88/TIR and NF-κB domains than QCT. In comparison, they demonstrated the greatest reduction in ulcer score and index, gastric MDA and nitric oxide (NO) contents, MyD88 and NF-κB expressions, and gastric TLR-4 immunostaining. They also enhanced the levels of GSH, CAT, COX-1, and COX-2 in the gastric mucosa, as well as HO-1 and Nrf2 expression, with histological regression in gastric mucosal lesions, with QPA-treated mice demonstrating the best GU healing. QPA is safe against all of the target organs and adverse pathways studied, with good ADME properties. However, further in vitro experiments are necessary to demonstrate the inhibitory effects of QPM and QPA on the protein targets of interest. In addition, preclinical research on its bioavailability and safety is essential before clinical management can be undertaken. Overall, the new QPA derivative could one day serve as the basis for a new class of potential antiulcer drugs.

An optimized denoised bias correction model with local pre-fitting function for weak boundary image segmentation

The active contour model (ACM) plays a paramount part in grasping visual properties of images and exacting targets of interest. It is overwhelming hardship for traditional ACMs to segment images with noise, intensity inhomogeneity or low contrast and consider computation speed for practical applicability. Therefore, an optimized denoised bias correction (ODBC) model incorporating the pre-piecewise fitting function and the variational denoised term into the energy function is proposed for images with low contrast and intensity inhomogeneity. An optimized gradient descent equation and an upgraded regularization term are produced to enhance the robustness and sensitivity of this model. Experiments are conducted to validate that ODBC model possesses the superiority of reliability and speed in segmenting images with inhomogeneous intensity and strengthens the robustness to initial contours. The results manifest that mIOU of ODBC model exceeds 0.9, superior to prime ACMs and deep learning schemes, in segmenting images associated and unrelated with the dataset.

Natural Killer Cell Infiltration in Prostate Cancers Predict Improved Patient Outcomes.

Natural killer (NK) cells are non-antigen specific innate immune cells that can be redirected to targets of interest using multiple strategies, although none are currently FDA-approved. We sought to evaluate NK cell infiltration into tumors to develop an improved understanding of which histologies may be most amenable to NK cell-based therapies currently in the developmental pipeline. DNA (targeted/whole-exome) and RNA (whole-transcriptome) sequencing was performed from tumors from 45 cancer types (N = 90,916 for all cancers and N = 3365 for prostate cancer) submitted to Caris Life Sciences. NK cell fractions and immune deconvolution were inferred from RNA-seq data using quanTIseq. Real-world overall survival (OS) and treatment status was determined and Kaplan-Meier estimates were calculated. Statistical significance was determined using X2 and Mann-Whitney U tests, with corrections for multiple comparisons where appropriate. In both a pan-tumor and prostate cancer (PCa)-specific setting, we demonstrated that NK cells represent a substantial proportion of the total cellular infiltrate (median range 2-9% for all tumors). Higher NK cell infiltration was associated with improved OS in 28 of 45 cancer types, including (PCa). NK cell infiltration was negatively correlated with common driver mutations and androgen receptor variants (AR-V7) in primary prostate biopsies, while positively correlated with negative immune regulators. Higher levels of NK cell infiltration were associated with patterns consistent with a compensatory anti-inflammatory response. Using the largest available dataset to date, we demonstrated that NK cells infiltrate a broad range of tumors, including both primary and metastatic PCa. NK cell infiltration is associated with improved PCa patient outcomes. This study demonstrates that NK cells are capable of trafficking to both primary and metastatic PCa and are a viable option for immunotherapy approaches moving forward. Future development of strategies to enhance tumor-infiltrating NK cell-mediated cytolytic activity and activation while limiting inhibitory pathways will be key.

Management of essential tremor deep brain stimulation-induced side effects.

Deep brain stimulation (DBS) is an effective surgical therapy for carefully selected patients with medication refractory essential tremor (ET). The most popular anatomical targets for ET DBS are the ventral intermedius nucleus (VIM) of the thalamus, the caudal zona incerta (cZI) and the posterior subthalamic area (PSA). Despite extensive knowledge in DBS programming for tremor suppression, it is not uncommon to experience stimulation induced side effects related to DBS therapy. Dysarthria, dysphagia, ataxia, and gait impairment are common stimulation induced side effects from modulation of brain tissue that surround the target of interest. In this review, we explore current evidence about the etiology of stimulation induced side effects in ET DBS and provide several evidence-based strategies to troubleshoot, reprogram and retain tremor suppression.

Post-clustering Prioritization Framework for Autonomous Decision Making in the Absence of Ground Truth via Hypothetical Probing

A generic prioritization framework is introduced for addressing the problem of automated prioritization of region of interest or target selection. The framework is based on the assumption that clustering of preliminary data for preidentified regions or targets of interest within an operational area has already occurred, i.e., post-classification, and that the clustering quality can be expressed as an energy/objective function. Region or target of interest prioritization then means to rank regions or targets of interest according to their probability of changing the energy/objective function value upon subsequent hypothetical probing as opposed to actually conducted reexamination, i.e., thorough follow-up or in-situ measurements. The mathematical formalism for calculating these probabilities to contribute to this change of the energy/objective function value is introduced and validated through numerical simulations. Moreover, these probabilities can also be understood as a confidence-check of the classification, i.e., the pre-clustering of the preliminary data. The operation of the prioritization framework is independent of the algorithm used to pre-cluster the preliminary data, and supports autonomous decision-making. It is widely applicable across many scientific disciplines and areas, ranging from the microscopic to the macroscopic scale. Due to its ability to help maximize scientific return while optimizing resource utilization, it is particularly relevant in the context of resource-constrained autonomous robotic planetary exploration as it may extend the Remaining Useful Lifetime (RUL) – a key aspect in Prognostics and Health Management (PHM) – of space missions. On a more general, PHM-relevant level, the prioritization framework may provide an additional mechanism of identifying and correcting the maintenance status of system components to assist predictive maintenance or condition-based maintenance.

Development and Comparison of 4-Thiouridine to Cytidine Base Conversion Reaction.

To study transcriptome dynamics without harming cells, it is essential to convert chemical bases. 4-Thiouridine (4sU) is a biocompatible uridine analogue that can be converted into a cytidine analogue. Although several reactions can convert 4sU into a cytidine analogue, few studies have compared the features of these reactions. In this study, we performed three reported base conversion reactions, including osmium tetroxide, iodoacetamide, and sodium periodate treatment, as well as a new reaction using 2,4-dinitrofluorobenzene. We compared the reaction time, conversion efficacy, and effects on reverse transcription. These reactions successfully converted 4sU into a cytidine analogue quantitatively using trinucleotides. However, the conversion efficacy and effect on reverse transcription vary depending on the reaction with the RNA transcript. OsO4 treatment followed by NH4Cl treatment showed the best base-conversion efficiency. Nevertheless, each reaction has its own advantages and disadvantages as a tool for studying the transcriptome. Therefore, it is crucial to select the appropriate reaction for the target of interest.

A targeted amplicon next-generation sequencing assay for tryptase genotyping to support personalized therapy in mast cell-related disorders.

Tryptase, the most abundant mast cell granule protein, is elevated in severe asthma patients independent of type 2 inflammation status. Higher active β tryptase allele counts are associated with higher levels of peripheral tryptase and lower clinical benefit from anti-IgE therapies. Tryptase is a therapeutic target of interest in severe asthma and chronic spontaneous urticaria. Active and inactive allele counts may enable stratification to assess response to therapies in asthmatic patient subpopulations. Tryptase gene loci TPSAB1 and TPSB2 have high levels of sequence identity, which makes genotyping a challenging task. Here, we report a targeted next-generation sequencing (NGS) assay and downstream bioinformatics analysis for determining polymorphisms at tryptase TPSAB1 and TPSB2 loci. Machine learning modeling using multiple polymorphisms in the tryptase loci was used to improve the accuracy of genotyping calls. The assay was tested and qualified on DNA extracted from whole blood of healthy donors and asthma patients, achieving accuracy of 96%, 96% and 94% for estimation of inactive α and βΙΙΙFS tryptase alleles and α duplication on TPSAB1, respectively. The reported NGS assay is a cost-effective method that is more efficient than Sanger sequencing and provides coverage to evaluate known as well as unreported tryptase polymorphisms.

Review of Advances in Coating and Functionalization of Gold Nanoparticles: From Theory to Biomedical Application.

Nanoparticles, especially gold nanoparticles (Au NPs) have gained increasing interest in biomedical applications. Used for disease prevention, diagnosis and therapies, its significant advantages in therapeutic efficacy and safety have been the main target of interest. Its application in immune system prevention, stability in physiological environments and cell membranes, low toxicity and optimal bioperformances are critical to the success of engineered nanomaterials. Its unique optical properties are great attractors. Recently, several physical and chemical methods for coating these NPs have been widely used. Biomolecules such as DNA, RNA, peptides, antibodies, proteins, carbohydrates and biopolymers, among others, have been widely used in coatings of Au NPs for various biomedical applications, thus increasing their biocompatibility while maintaining their biological functions. This review mainly presents a general and representative view of the different types of coatings and Au NP functionalization using various biomolecules, strategies and functionalization mechanisms.

Metals in Cancer Research: Beyond Platinum Metallodrugs.

The discovery of the medicinal properties of platinum complexes has fueled the design and synthesis of new anticancer metallodrugs endowed with unique modes of action (MoA). Among the various families of experimental antiproliferative agents, organometallics have emerged as ideal platforms to control the compounds' reactivity and stability in a physiological environment. This is advantageous to efficiently deliver novel prodrug activation strategies, as well as to design metallodrugs acting only via noncovalent interactions with their pharmacological targets. Noteworthy, another justification for the advance of organometallic compounds for therapy stems from their ability to catalyze bioorthogonal reactions in cancer cells. When not yet ideal as drug leads, such compounds can be used as selective chemical tools that benefit from the advantages of catalytic amplification to either label the target of interest (e.g., proteins) or boost the output of biochemical signals. Examples of metallodrugs for the so-called "catalysis in cells" are considered in this Outlook together with other organometallic drug candidates. The selected case studies are discussed in the frame of more general challenges in the field of medicinal inorganic chemistry.

Relevance of detection of RAF fusion transcripts in pan-negative melanoma in routine practice.

Pan-negative melanomas account for 30% of melanomas. In case of immunotherapy failure, therapeutic options are limited. Oncogene fusions represent a target of interest in many solid cancers. In melanoma, the frequency of oncogene fusion is not well documented and not routinely investigated. We conducted a single-center retrospective study. The objective was to determine the frequency of oncogene fusion detected by RNA sequencing, in patients with advanced or metastatic pan-negative melanoma. In parallel, an extended molecular alteration search was performed using extended targeted next-generation sequencing. We identified 59 patients with advanced pan-negative melanoma between January 2021 and January 2023. It was a cutaneous melanoma in 71.1% of the cases, a mucous melanoma in 15.2% of the cases. We identified nine patients with a RAF fusion, including seven BRAF gene fusion and two RAF1 fusion. Of the other molecular alterations, NF1 mutation was the most frequent molecular alteration identified. Among the nine patients with RAF fusions, all the patients initially received treatment with anti-PD1 ± anti-CTLA4 immunotherapy. After immunotherapy failure, five patients benefited from second-line targeted therapy (two with BRAF and MEK inhibitors combination, three MEK inhibitors alone). The response rate was 20%. In a population of pan-negative melanoma, we detected 15.2% of RAF fusion. Fusion detection allowed the introduction of a second line of targeted therapy, in the absence of a validated therapeutic option in 55.5% of cases. This study suggests the relevance of detecting RAF fusion in a selected population.

Transcriptome analysis of salivary glands of rabies-virus-infected mice.

Rabies is a fatal zoonotic disease that poses a threat to public health. Rabies virus (RABV) is excreted in the saliva of infected animals, and is primarily transmitted by bite. The role of the salivary glands in virus propagation is significant, but has been less studied in the pathogenic mechanisms of RABV. To identify functionally important genes in the salivary glands, we used RNA sequencing (RNA-seq) to establish and analyze mRNA expression profiles in parotid tissue infected with two RABV strains, CVS-11 and PB4. The biological functions of differentially expressed genes (DEGs) were determined by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, which revealed 3,764 DEGs (678 up-regulated and 3,086 down-regulated) in the CVS-11 infected group and 4,557 DEGs (874 up-regulated and 3,683 down-regulated) in the PB4 infected group. Various biological processes are involved, including the salivary secretion pathway and the phosphatidylinositol 3-kinase-Akt (PI3K-Akt) signaling pathway. This study provides the first mapping of the transcriptome changes in response to RABV infection in parotid tissue, offering new insights into the study of RABV-affected salivary gland function and RABV pathogenic mechanisms in parotid tissue. The salivary gland-enriched transcripts may be potential targets of interest for rabies disease control.

Quantifying the Transit Light Source Effect: Measurements of Spot Temperature and Coverage on the Photosphere of AU Microscopii with High-resolution Spectroscopy and Multicolor Photometry

AU Microscopii (AU Mic) is an active 24 ± 3 Myr pre-main-sequence M dwarf in the stellar neighborhood (d = 9.7 pc) with a rotation period of 4.86 days. The two transiting planets orbiting AU Mic, AU Mic b and c, are warm sub-Neptunes on 8.5 and 18.9 day periods and are targets of interest for atmospheric observations of young planets. Here we study AU Mic’s unocculted starspots using ground-based photometry and spectra in order to complement current and future transmission spectroscopy of its planets. We gathered multicolor Las Cumbres Observatory (LCO) 0.4 m SBIG photometry to study the star's rotational modulations and LCO Network of Robotic Echelle Spectrographs high-resolution spectra to measure the different spectral components within the integrated spectrum of the star, parameterized by three spectral components and their coverage fractions. We find AU Mic’s surface has at least two spectral components: a T amb = 4003−14+15 K ambient photosphere and cool spots that have a temperature of T spot = 3003−71+63 K, covering a globally averaged area of 39% ± 4% which increases and decreases by 5.1% ± 0.3% from the average throughout a rotation. We also detect a third flux component with a filling factor less than 0.5% and a largely uncertain temperature between 8500 and 10,000 K that we attribute to flare flux not entirely omitted when time averaging the spectra. We include measurements of spot characteristics using a two-temperature model, which we find agree strongly with the three-temperature results. Our expanded use of various techniques to study starspots will help us better understand this system and may have applications for interpreting the transmission spectra for exoplanets transiting stars of a wide range of activity levels.

Brain Shift during Staged Deep Brain Stimulation for Movement Disorders

Introduction: Deep brain stimulation (DBS) is a routine neurosurgical procedure utilized to treat various movement disorders including Parkinson’s disease (PD), essential tremor (ET), and dystonia. Treatment efficacy is dependent on stereotactic accuracy of lead placement into the deep brain target of interest. However, brain shift attributed to pneumocephalus can introduce unpredictable inaccuracies during DBS lead placement. This study aimed to determine whether intracranial air is associated with brain shift in patients undergoing staged DBS surgery. Methods: We retrospectively evaluated 46 patients who underwent staged DBS surgery for PD, ET, and dystonia. Due to the staged nature of DBS surgery at our institution, the first electrode placement is used as a concrete fiducial marker for movement in the target location. Postoperative computed tomography (CT) images after the first electrode implantation, as well as preoperative, and postoperative CT images after the second electrode implantation were collected. Images were analyzed in stereotactic targeting software (BrainLab); intracranial air was manually segmented, and electrode shift was measured in the x, y, and z plane, as well as a Euclidian distance on each set of merged CT scans. A Pearson correlation analysis was used to determine the relationship between intracranial air and brain shift, and student’s t test was used to compare means between patients with and without radiographic evidence of intracranial air. Results: Thirty-six patients had pneumocephalus after the first electrode implantation, while 35 had pneumocephalus after the second electrode implantation. Accumulation of intracranial air following the first electrode implantation (4.49 ± 6.05 cm³) was significantly correlated with brain shift along the y axis (0.04 ± 0.35 mm; r (34) = 0.36; p = 0.03), as well as the Euclidean distance of deviation (0.57 ± 0.33 mm; r (34) = 0.33; p = 0.05) indicating statistically significant shift on the ipsilateral side. However, there was no significant correlation between intracranial air and brain shift following the second electrode implantation, suggesting contralateral shift is minimal. Furthermore, there was no significant difference in brain shift between patients with and without radiographic evidence of intracranial air following both electrode implantation surgeries. Conclusion: Despite observing volumes as high as 22.0 cm³ in patients with radiographic evidence of pneumocephalus, there was no significant difference in brain shift when compared to patients without pneumocephalus. Furthermore, the mean magnitude of brain shift was <1.0 mm regardless of whether pneumocephalus was presenting, suggesting that intracranial air accumulation may not produce clinical significant brain shift in our patients.

Nanobodies: a promising approach to treatment of viral diseases.

Since their discovery in the 1990s, heavy chain antibodies have garnered significant interest in the scientific community. These antibodies, found in camelids such as llamas and alpacas, exhibit distinct characteristics from conventional antibodies due to the absence of a light chain in their structure. Furthermore, they possess a single antigen-binding domain known as VHH or Nanobody (Nb). With a small size of approximately 15 kDa, these Nbs demonstrate improved characteristics compared to conventional antibodies, including greater physicochemical stability and enhanced biodistribution, enabling them to bind inaccessible epitopes more effectively. As a result, Nbs have found numerous applications in various medical and veterinary fields, particularly in diagnostics and therapeutics. Advances in biotechnology have made the production of recombinant antibodies feasible and compatible with large-scale manufacturing. Through the construction of immune phage libraries that display VHHs and subsequent selection through biopanning, it has become possible to isolate specific Nbs targeting pharmaceutical targets of interest, such as viruses. This review describes the processes involved in nanobody production, from hyperimmunization to purification, with the aim of their application in the pharmaceutical industry.