The Ratio of Remaining to Expected Small Bowel Length Predicts Enteral Autonomy in Pediatric Patients with Short Bowel Syndrome.

BACKGROUND: Pediatric patients with short bowel syndrome (SBS) often require long-term parenteral nutrition and intravenous fluid support (PN) until enteral autonomy (EA). However, long-term PN accounts for many complications. We aimed to investigate the outcome and predictors of EA in these patients. MATERIAL AND METHODS: This retrospective observational study was conducted in Children's Medical Center, Chang Gung Memorial Hospital, a tertiary hospital in Northern Taiwan. Twenty-four patients afflicted with short bowel syndrome between 2002 and 2021 were included. Demographics, operation results, follow-up status, complications, and outcomes were reviewed. RESULTS: Among the 24 patients, 14 were males (58%). The median age at bowel resection was 3 days (IQR, 1.3 to 28.8 days). The most common etiologies were total/subtotal intestinal aganglionosis (TIA) (N=6) and malrotation with midgut volvulus (N=6). The median length of the residual small intestine was 25cm (IQR, 7.8 to 71.3cm). Ten (41.7%) had preserved ileocecal valve, and 14 (58.3%) had colon-in-continuity. Intestinal failure-associated liver disease (IFALD) occurred in 14 patients (58.3%), but none had advanced disease. Seven (29.2%) patients achieved enteral autonomy after 10.1±7.3 months. Five patients (21%) expired due to sepsis. Logistic regression and Kaplan-Meier analysis showed the predictors of enteral autonomy were remaining-to-expected small bowel length ratio > 25% and the absence of IFALD. CONCLUSIONS: In this pediatric short bowel syndrome study, enteral autonomy was achieved in 29% after a mean PN duration of 10 months. The remaining-to-expected small bowel length ratio at bowel resection was the most critical predictor of enteral autonomy.

Pulmonary thromboembolism: a case report and misdiagnosis analysis of a 63-year-old female patient.

This paper presents a case of a 63-year-old female patient who was initially misdiagnosed with mycoplasma pneumonia due to symptoms such as chest pain, hemoptysis, and fever, but was later confirmed to have pulmonary thromboembolism (PTE) through further examination. This case highlights the similarities between PTE and pneumonia in terms of symptoms, as well as the complexity of PTE diagnosis. The article provides a detailed description of the patient's medical history, symptoms, examination process, and treatment outcomes. Furthermore, it discusses the possible reasons for the misdiagnosis, including insufficient awareness of PTE among physicians, lack of in-depth investigation into the causes of abnormally elevated D-dimer levels, the non-specific clinical manifestations of PTE, and the concerns of the patient's family regarding pulmonary artery CTA examination. Additionally, the article emphasizes the importance of clinicians in improving their ability to differentiate and diagnose PTE, rationally utilizing clinical examination methods, and ensuring timely diagnosis and treatment of PTE.

Prevalence and risk factors of osteosarcopenia in elderly patients with uncontrolled type 2 diabetes.

PURPOSE: To assess the prevalence of osteosarcopenia (OS) in elderly patients with type 2 diabetes mellitus (T2DM) and explore the related risk factors for developing this condition. METHODS: This cross-sectional study enrolled hospitalized T2DM patients aged 60 years and older. Patients underwent assessments of total hip bone mineral density (BMD), grip strength, the Short Physical Performance Battery (SPPB), and body composition. Based on the 2019 Asian Working Group for Sarcopenia (AWGS) criteria, appendicular skeletal muscle mass (ASM), grip strength, and SPPB were measured to diagnose sarcopenia. BMD and T values of the lumbar spine and hip were measured using dual-energy X-ray absorptiometry (DXA). Osteosarcopenia was defined when both sarcopenia and osteoporosis criteria were met. Statistical analysis included binary logistic regression to identify significant risk factors. RESULTS: A total of 254 hospitalized T2DM patients (80 males and 174 females) were included. They were divided into T2DM-OS (n = 58) and T2DM-NOS (n = 196) groups based on the presence of osteosarcopenia. The average ages were 72.724 ± 6.463 and 69.265 ± 6.035 years, respectively. The prevalence of osteosarcopenia in T2DM patients was 22.8%, with 20.7% (12 males) and 79.3% (46 females) in the T2DM-OS group. After adjusting for confounding factors, it was found that male gender (OR: 5.738, 95% CI: 1.602-20.551, P = 0.007), fasting plasma glucose (OR: 0.904, 95% CI: 0.821-0.995, P = 0.038), and ASMI (OR: 0.049, 95% CI: 0.013-0.184, P < 0.001) were major influencing factors for the development of osteosarcopenia in elderly T2DM patients. CONCLUSIONS: The prevalence of T2DM-OS is relatively high, with male gender, low fasting plasma glucose, and low ASMI identified as risk factors.

CCN1 Is a Therapeutic Target for Reperfused Ischemic Brain Injury.

Ischemic stroke can lead to systemic inflammation, which can activate peripheral immune cells, causing neuroinflammation and brain injury. Meningeal lymphatics play a crucial role in transporting solutes and immune cells out of the brain and draining them into cervical lymph nodes (CLNs). However, the role of meningeal lymphatics in regulating systemic inflammation during the reperfusion stage after ischemia is not well understood. In this study, we demonstrated that brain infarct size, neuronal loss, and the effector function of inflammatory macrophage subsets were reduced after ischemia-reperfusion and disruption of meningeal lymphatics. Spatial memory function was improved in the late stage of ischemic stroke following meningeal lymphatic disruption. Brain-infiltrating immune cells, including neutrophils, monocytes, and T and natural killer cells, were reduced after cerebral ischemia-reperfusion and meningeal lymphatic disruption. Single-cell RNA sequencing analysis revealed that meningeal lymphatic disruption reprogrammed the transcriptome profile related to chemotaxis and leukocyte migration in CLN lymphatic endothelial cells (LECs), and it also decreased chemotactic CCN1 expression in floor LECs. Replenishment of CCN1 through intraventricular injection increased brain infarct size and neuronal loss, while restoring numbers of macrophages/microglia in the brains of meningeal lymphatic-disrupted mice after ischemic stroke. Blocking CCN1 in cerebrospinal fluid reduced brain infarcts and improves spatial memory function after ischemia-reperfusion injury. In summary, this study indicates that CCN1-mediated detrimental inflammation was alleviated after cerebral ischemia-reperfusion injury and meningeal lymphatic disruption. CCN1 represents a novel therapeutic target for inhibiting systemic inflammation in the brain-CLN axis after ischemia-reperfusion injury.

Efficacy of probiotics in the treatment of oral mucositis in head and neck cancer patients: A systematic review and meta-analysis.

OBJECTIVES: To assess the effect of probiotics in oral mucositis induced by chemotherapy or radiotherapy on patients with head and neck cancer (HNC). METHODS: The PubMed, Embase, Cochrane Library, Clinical trials were screened from January 2010 to April 2024. Randomized clinical trials (RCTs) comparing the efficacy of probiotics in treatment of oral mucositis in HNC were eligible. Outcomes of interest were incidence of oral mucositis and severe oral mucositis. The PROSPERO registration number was 42 022 384 685. The Cochrane risk-of-bias tool (RoB2) was used to assess methodological quality of studies and GRADE criteria (GRADEpro) was applied for rating the certainty of evidence. Meta-analysis was performed by using RevMan 5.4. RESULTS: A total of eight RCTs comprising 691 patients with HNC were included in this meta-analysis. Probiotics administration significantly reduced the incidence of SOM (RR = 0.60, 95%CI: 0.46-0.78, P = 0.0002). However, it showed no distinct advantage in reducing the overall incidence of oral mucositis (RR = 0.88, 95%CI: 0.76-1.02, P = 0.08). Subgroup analysis found more benefit for reducing SOM in multi-bacterial treated group (RR = 0.35, 95%CI: 0.17-0.73, P = 0.005) than mono-bacterial treated group (RR = 0.69, 95%CI: 0.58-0.82, P < 0.0001). In Addition, probiotics could reduce the incidence of SOM in nasopharyngeal carcinoma (NPC) treated with intensity-modulated radiotherapy (RR = 0.43, 95%CI: 0.26-0.70, P = 0.0006). CONCLUSION: Probiotics reduced the incidence of SOM caused by chemotherapy or radiotherapy for HNC. The multi-bacterial combination therapy was more efficacious than the mono-bacterial therapy. Moreover, probiotics also reduced the incidence of SOM in nasopharyngeal carcinoma. However, the advantage of probiotics had not been established in the overall incidence of OM.

A review of in vitro stochastic and non-stochastic affinity maturation strategies for phage display derived monoclonal antibodies.

Monoclonal antibodies identified using display technologies like phage display occasionally suffers from a lack of affinity making it unsuitable for application. This drawback is circumvented with the application of affinity maturation. Affinity maturation is an essential step in the natural evolution of antibodies in the immune system. The evolution of molecular based methods has seen the development of various mutagenesis approaches. This allows for the natural evolutionary process during somatic hypermutation to be replicated in the laboratories for affinity maturation to fine-tune the affinity and selectivity of antibodies. In this review, we will discuss affinity maturation strategies for mAbs generated through phage display systems. The review will highlight various in vitro stochastic and non-stochastic affinity maturation approaches that includes but are not limited to random mutagenesis, site-directed mutagenesis, and gene synthesis.

High percentage of bone marrow CD8+ tissue-resident-like memory T cells predicts inferior survival in patients with acute myeloid leukemia.

Tissue-resident memory T (TRM) cells infiltrating solid tumors could influence tumor progression and the response to immune therapies. However, the proportion and prognostic value of TRM cells in the bone marrow (BM) of patients with acute myeloid leukemia (AML) are unclear. In this study, we used flow cytometry to assay the phenotype of 49 BM samples from patients newly diagnosed with AML (ND-AML). We found that the BM CD8+ effector memory (TEM) cells highly expressed CD69 (CD8+ TRM-like T cells), and their percentage was significantly increased in patients with ND-AML compared with that in healthy individuals (HI). The high percentage of CD8+ TRM-like subset was associated with poor overall survival in our ND-AML cohort. The Kaplan-Meier Plotter database verified a significantly reduced survival rate among patients with high expression of CD8+ TRM-like T cell characteristic genes (CD8A, CD69, and TOX), especially the M4 and M5 subtypes. Phenotypic analysis revealed that the BM CD8+ TRM-like subpopulation exhibited exhausted T cell characteristics, but its high expression of CD27 and CD28 and low expression of CD57 suggested its high proliferative potential. The single-cell proteogenomic dataset confirmed the existence of TRM-like CD8+ T cells in the BM of patients with AML and verified the high expression of immune checkpoints and costimulatory molecules. In conclusion, we found that the accumulation of BM CD8+ TRM-like cells could be an immune-related survival prediction marker for patients with AML.

Lactiplantibacillus plantarum RS20D Alleviates Male Reproductive Toxicity Induced by Pubertal Exposure to Di-n-butyl Phthalate and Mono-n-butyl Phthalate.

Phthalate acid esters (PAEs) and their metabolites, such as di-n-butyl phthalate (DBP) and mono-n-butyl phthalate (MBP), are known to cause male reproductive damage. Lactiplantibacillus plantarum RS20D has demonstrated the ability to remove both DBP and MBP in vitro, suggesting its potential as a detoxifying agent against these compounds. This study aimed to investigate the protective effects of RS20D on DBP or MBP-induced male reproductive toxicity in adolescent rats. Oral administration of RS20D significantly mitigated the histological damage to the testes caused by MBP or DBP, restored sperm concentration, morphological abnormalities, and the proliferation index in MBP-exposed rats, and partially reversed spermatogenic damage in DBP-exposed rats. Furthermore, RS20D restored serum levels of estradiol (E2) and testosterone, and superoxide dismutase (SOD) activity in DBP-exposed rats, significantly increased testosterone levels in MBP-exposed rats, and restored copper (Cu) concentrations in the testes after exposure to DBP or MBP. Additionally, RS20D effectively modulated the intestinal microbiota in DBP-exposed rats and partially ameliorated dysbiosis induced by MBP, which may be associated with the alleviation of reproductive toxic effects induced by DBP or MBP. In conclusion, this study demonstrates that RS20D administration can alleviate male reproductive toxicity and gut dysbacteriosis induced by DBP or MBP exposure, providing a dietary strategy for the bioremediation of PAEs and their metabolites.

Influence of the community assemblage on sulfur distributions in the South China sea.

Marine distribution of dimethylsulfoniopropionate (DMSP) and its cleavage product dimethyl sulfide (DMS) is greatly affected by the community structures of bacteria, phytoplankton, and zooplankton. Spatial distributions of dissolved and particulate DMSP (DMSPd,p), and DMS were measured and their relationships with DMSP lyase activity (DLA), abundance of DMSP-consuming bacteria (DCB), and the community structures of phytoplankton, zooplankton, and bacteria were determined during summer in the South China Sea (SCS). The depth distributions of DMSPd,p exhibited a similar trend with Chl a, reaching their maxima in the mixing layer. The DMS concentration was positively correlated with DCB abundance and DLA, indicating that DCB and DMSP lyase had a significant effect on DMS production. High DMS concentrations in the horizontal distribution coincided with high DCB abundance and DLA and may be due to the rapid growth of phytoplankton resulting from the high dissolved inorganic nitrogen concentration brought by the cold vortices. Moreover, the highest copepod abundance at station G3 coincided with the highest DMS concentrations there among stations B4, F2, and G3. These results suggest that copepod may play an important role in DMS production. The bacterial SAR11 clade was positively correlated with DLA, indicating its significant contribution to DMSP degradation in the SCS. These findings contribute to the understanding of the effect of the community assemblage on DMSP/DMS distributions in the SCS dominated by mesoscale vortices.

Generation of a Naïve Human scFv Phage Display Library and Panning Selection.

Phage display antibody libraries have been successfully used as the essential tool to produce monoclonal antibodies against a plethora of targets ranging from diseases to native biologically important proteins as well as small molecules. It is well documented that diverse antibody genes are the major genetic source for the construction of a high-quality antibody library and selection of high-affinity antibodies. Naïve antibody libraries are derived using the IgM repertoire of healthy donors obtained from B-cells isolated from human peripheral blood mononuclear cell (PBMC). Single-chain fragment variable (scFv) is a routinely used format due to its smaller size and preference for phage display. The process involves the use of a two-step cloning method for library construction. The protocol also covers the biopanning process for target positive clone selection.

Development and internal validation of a nomogram based on peripheral blood inflammatory markers for predicting prognosis in nasopharyngeal carcinoma.

BACKGROUND: Inflammatory markers, including the product of neutrophil count, platelet count, and monocyte count divided by lymphocyte count (PIV) and the platelet-to-white blood cell ratio (PWR), have not been previously reported as prognostic factors in nasopharyngeal carcinoma (NPC) patients. In order to predict overall survival (OS) in NPC patients, our goal was to create and internally evaluate a nomogram based on inflammatory markers (PIV, PWR). METHODS: A retrospective study was done on patients who received an NPC diagnosis between January 2015 and December 2018. After identifying independent prognostic indicators linked to OS using Cox proportional hazards regression analysis, we created a nomogram with the factors we had chosen. RESULTS: A total of 630 NPC patients in all were split into training (n = 441) and validation sets (n = 189) after being enrolled in a population-based study in 2015-2018 and monitored for a median of 5.9 years. In the training set, the age, PIV, and PWR, selected as independent predictors for OS via multivariate Cox's regression model, were chosen to develop a nomogram. Both training and validation cohorts had C-indices of 0.850 (95% confidence interval [CI]: 0.768-0.849) and 0.851 (95% CI: 0.765-0.877). Furthermore, compared with traditional TNM staging, our nomogram demonstrated greater accuracy in predicting patient outcomes. The risk stratification model derived from our prediction model may facilitate personalized treatment strategies for NPC patients. CONCLUSION: Our findings confirmed the prognostic significance of the PWR and PIV in NPC. High PIV levels (>363.47) and low PWR (≤36.42) values are associated with worse OS in NPC patients.

Inhibition of NRF2 enhances the acute myeloid leukemia cell death induced by venetoclax via the ferroptosis pathway.

Venetoclax, an inhibitor that selectively targets B cell lymphoma-2 (BCL-2) that has been approved for treating adult acute myeloid leukemia (AML) in combination with hypomethylating agents. However, its short duration of response and emergence of resistance are significant issues. In this study, we found that the sensitivity of AML cells to venetoclax was considerably enhanced by ML385, an inhibitor of the ferroptosis factor nuclear transcription factor erythroid 2-related factor 2 (NRF2). Using AML samples, we verified that NRF2 and its target gene ferritin heavy chain 1 (FTH1) were highly expressed in patients with AML and correlated with poor prognosis. Downregulation of NRF2 could inhibit FTH1 expression and significantly enhance the venetoclax-induced labile iron pool and lipid peroxidation. By contrast, NRF2 overexpression or administration of the reactive oxygen species inhibitor N-acetylcysteine and vitamin E could effectively suppress the anti-AML effects of ML385+venetoclax. Furthermore, the ferroptosis inducer erastin increased the anti-AML effects of venetoclax. Our study demonstrated that NRF2 inhibition could enhance the AML cell death induced by venetoclax via the ferroptosis pathway. Thus, the combination of ML385 with venetoclax may offer a favorable strategy for AML treatment.

Orientation of Thiocyanate Ions Tuning the Electron-Phonon Interactions in Pseudohalide Perovskites.

Although the importance of electron-phonon interactions on the optoelectronic properties of perovskites has been well documented, the structural origin of electron-phonon interactions remains largely unexplored. In this study, using pseudohalide perovskites Cs2Pb(SCN)2I2(1-x)Br2x as a model, we have revealed how the orientation of SCN- anions tunes the electron-phonon interactions and the effective charge-carrier mobility by utilizing femtosecond sum frequency generation vibrational spectroscopy, supplemented by photoluminescence spectroscopy and femtosecond optical-pump terahertz-probe spectroscopy. The coupling between neighboring SCN- anions decreases as the Br content (x) increases but does not have a significant effect on the electron-phonon interactions. In contrast, the orientation angle of SCN- anions has a strong correlation with the electron-phonon interaction and effective charge-carrier mobility, that is, a more parallel orientation of SCN- anions leads to a higher electron-phonon interaction and lower effective charge-carrier mobility. This finding provides a molecule-level understanding of the inorganic lattice structure in tuning electron-phonon interactions and may offer valuable guidance for optimizing the optoelectronic properties of perovskites.

Spacer Engineering Enables Fine-Tuned Thin Film Microstructure and Efficient Charge Transport for Ultrasensitive 2D Perovskite-Based Heterojunction Phototransistors and Optoelectronic Synapses.

2D Ruddlesden-Popper phase layered perovskites (RPLPs) hold great promise for optoelectronic applications. In this study, a series of high-performance heterojunction phototransistors (HPTs) based on RPLPs with different organic spacer cations (namely butylammonium (BA+), cyclohexylammonium (CyHA+), phenethylammonium (PEA+), p-fluorophenylethylammonium (p-F-PEA+), and 2-thiophenethylammonium (2-ThEA+)) are fabricated successfully, in which high-mobility organic semiconductor 2,7-dioctyl[1]benzothieno[3,2-b]benzothiophene is adopted to form type II heterojunction channels with RPLPs. The 2-ThEA+-RPLP-based HPTs show the highest photosensitivity of 3.18 × 107 and the best detectivity of 9.00 × 1018 Jones, while the p-F-PEA+-RPLP-based ones exhibit the highest photoresponsivity of 5.51 × 106 A W-1 and external quantum efficiency of 1.32 × 109%, all of which are among the highest reported values to date. These heterojunction systems also mimicked several optically controllable fundamental characteristics of biological synapses, including excitatory postsynaptic current, paired-pulse facilitation, and the transition from short-term memory to long-term memory states. The device based on 2-ThEA+-RPLP film shows an ultra-high PPF index of 234%. Moreover, spacer engineering brought fine-tuned thin film microstructures and efficient charge transport/transfer, which contributes to the superior photodetection performance and synaptic functions of these RPLP-based HPTs. In-depth structure-property correlations between the organic spacer cations/RPLPs and thin film microstructure/device performance are systematically investigated.

Colon-Accumulated Gold Nanoclusters Alleviate Intestinal Inflammation and Prevent Secondary Colorectal Carcinogenesis via Nrf2-Dependent Macrophage Reprogramming.

Inflammatory bowel disease (IBD) is one of the main factors leading to colitis-associated colorectal cancer (CAC). Therefore, it is critical to develop an effective treatment for IBD to prevent secondary colorectal carcinogenesis. M2 macrophages play crucial roles in the resolution phase of intestinal inflammation. However, traditional drugs rarely target intestinal M2 macrophages, and they are not easily cleared. Gold nanoclusters are known for their in vivo safety and intrinsic biomedical activities. In this study, a glutathione-protected gold nanocluster is synthesized and evaluated, namely, GA. Interestingly, GA specifically accumulates in the colon during IBD. Furthermore, GA not only promotes M2 differentiation of IL-4-treated peritoneal macrophages but also reprograms macrophage polarization from M1 to M2 in a pro-inflammatory environment. Mechanistically, this regulatory effect is exerted through activating the antioxidant Nrf2 signaling pathway, but not traditional STAT6. When applied in IBD mice, we found that GA elevates M2 macrophages and alleviates IBD in an Nrf2-dependent manner, evidenced by the abolished therapeutic effect upon Nrf2 inhibitor treatment. Most importantly, GA administration significantly suppresses AOM/DSS-induced CAC, without causing obvious tissue damage, providing critical evidence for the potential application of gold nanoclusters as nanomedicine for the treatment of IBD and CAC.

Construction of Naïve and Immune Human Fab Phage Display Library.

Phage display has been applied successfully for the rapid isolation of monoclonal antibodies against various targets including infectious diseases, autoantigens, cancer markers, and even small molecules. The main component in any phage display experiment is the availability of an antibody library to carry out the selection process of target-specific antibodies through an iterative process termed as biopanning. To generate human antibody libraries, the antibody repertoire can be obtained from human peripheral blood mononuclear cell (PBMC) or directly from cell-sorted B-cell populations. The choice of antibody isotype is dictated by the nature of the library. Naïve libraries would utilize IgM repertoires, whereas the IgG repertoire is commonly used for immune libraries. Antibody genes are amplified through polymerase chain reaction (PCR) and paired in a combinatorial fashion to expand the diversity of the cloned library repertoire. The protocol here describes the use of a two-step cloning method that can be applied for the construction of either a naïve or immune human antibody library in Fab format followed by the subsequent panning.

Antibody Phage Display.

The application of antibodies has transcended across many areas of work but mainly as a research tool, for diagnostic and for therapeutic applications. Antibodies are immunoproteins from vertebrates that have the unique property of specifically binding foreign molecules and distinguish target antigens. This property allows antibodies to effectively protect the host from infections. Apart from the hybridoma technology using transgenic animals, antibody phage display is commonly considered the gold standard technique for the isolation of human monoclonal antibodies. The concept of antibody phage display surrounds the ability to display antibody fragments on the surface of M13 bacteriophage particles with the corresponding gene packaged within the particle. A repetitive in vitro affinity based selection process permits the enrichment of target specific binders. This process of recombinant human monoclonal antibody generation also enables additional engineering for various applications. This makes phage display an indispensable technique for antibody development and engineering activities.

Streptavidin-Coated Solid-Phase Extraction (SPE) Tips for Antibody Phage Display Biopanning.

Phage display is a technique that allows the presentation of unique proteins on the surface of bacteriophages. The phage particles are usually screened via repetitive rounds of antigen-guided selection and phage amplification. The main advantage of this approach lies in the physical linkage between phenotype and genotype. This feature allows the isolation of single unique clones from a panning campaign consisting of a highly diverse population of clones. Due to the high-throughput nature of this technique, different approaches have been developed to assist phage display selections. One of which involves utilizing a streptavidin-coated solid-phase extraction (SPE) tip that is mounted to an electronically controlled motorized multichannel pipette. In this chapter, we will entail the procedures involved in the adaptation of a commercial SPE tip (MSIA™ streptavidin D.A.R.T's®) as the solid phase. This protocol is an updated version of a previous protocol with some minor refinements.

Subtractive panning for the isolation of monoclonal PEPITEM peptide antibody by phage display.

Antibody phage display is a key tool for the development of monoclonal antibodies against various targets. However, the development of anti-peptide antibodies is a challenging process due to the small size of peptides for binding. This makes anchoring of peptides a preferred approach for panning experiments. A common approach is by using streptavidin as the anchor protein to present biotinylated peptides for panning. Here, we propose the use of recombinant expression of the target peptide and an immunogenic protein as a fusion for panning. The peptide inhibitor of trans-endothelial migration (PEPITEM) peptide sequence was fused to the Mycobacterium tuberculosis (Mtb) α-crystalline (AC) as an anchor protein. The panning process was carried out by subtractive selection of the antibody library against the AC protein first, followed by binding to the library to PEPITEM fused AC (PEPI-AC). A unique monoclonal scFv antibodies with good specificity were identified. In conclusion, the use of an alternative anchor protein to present the peptide sequence coupled with subtractive panning allows for the identification of unique monoclonal antibodies against a peptide target.