Shay Bracha - Academia.edu (original) (raw)

Papers by Shay Bracha

Cell Stress and Chaperones, 2022

Table S1. Nucleotide sequences of specific PCR primers. (PDF 336 kb)

Figure S1. Comparison of nucleotide sequences of kiss1 (A) and amino acid sequences of Kiss-1 (B)... more Figure S1. Comparison of nucleotide sequences of kiss1 (A) and amino acid sequences of Kiss-1 (B) in the dog, human, and mouse. (PDF 462 kb)

Analytical and Bioanalytical Chemistry, 2021

Separation of blood plasma or serum from blood is essential for accurate analysis. Conventional b... more Separation of blood plasma or serum from blood is essential for accurate analysis. Conventional blood separation requires instrumentation, reagents, and large sample volumes, limiting this process to laboratory environments with trained personnel. Full implementation of effective blood separation and analysis on microliter sample volumes for point of care (POC) diagnostics has proven extremely challenging resulting in a growing market demand, with common challenges such as expensive device fabrication processes or devices being comprised of materials which are not easily disposable. We developed a membrane-based wicking microfluidic device which is made using a simple fabrication process. This device uses a unique 3D flow channel geometry, fabricated in a polycaprolactone-filled glass microfiber membrane, to efficiently separate microliter sample volumes of blood. Colorimetric assay chemistries were integrated to demonstrate utility of these devices in POC diagnostics. The devices are capable of separating both fresh and anticoagulant-treated blood at microscale sample volumes (<15.0 μL). Modifications to the base device are also reported herein which increased sample volume capacity and separation efficiency. Integrated colorimetric assay enabled semi-quantitative detection of conjugated bilirubin in real blood samples (1.0–1.5 mg/dL). These blood separation devices, fabricated on polycaprolactone-filled glass microfiber, enabled effective blood plasma (anticoagulant-treated blood) and serum (fresh blood) separation with microscale sample volumes. Sample volume capacity and separation efficiency are customizable for specific applications and devices can be integrated with downstream assay chemistries to develop complete POC devices that offer blood separation and diagnostics at the same time on a single membrane.

Journal of the American Animal Hospital Association, 2009

A 9-year-old Bouvier des Flandres was presented with coughing, lethargy, chylous pleural effusion... more A 9-year-old Bouvier des Flandres was presented with coughing, lethargy, chylous pleural effusion, and a heart murmur. An echocardiogram revealed the presence of an intracardiac mass causing right ventricular outflow tract obstruction. The mass was successfully removed surgically, using total inflow occlusion. Histopathology and immunohistochemistry identified the tumor as an ectopic thyroid carcinoma. The dog was euthanized 11 months after diagnosis at the request of the owner because of nonresolving chylothorax.

Pediatric Blood & Cancer, 2013

Journal of Small Animal Practice, 2014

To evaluate the tolerability of a piroxicam and cyclophosphamide metronomic treatment protocol co... more To evaluate the tolerability of a piroxicam and cyclophosphamide metronomic treatment protocol combined with carboplatin alone or carboplatin and doxorubicin at maximal-tolerated doses. Retrospective study of 30 dogs diagnosed with osteosarcoma. All dogs underwent amputation and chemotherapy treatment with one of the two maximal-tolerated dose protocols. Metronomic chemotherapy was administered in conjunction with these protocols, and continued subsequently. The protocols included 0 · 3 mg/kg piroxicam and 10 to 12 mg/M(2) cyclophosphamide with 300 mg/M(2) carboplatin alone, or 300 mg/M(2) carboplatin alternating with 30 mg/M(2) doxorubicin. Fourteen dogs were treated with the carboplatin and metronomic protocol and 16 were treated with the carboplatin alternating with doxorubicin and metronomic protocol. Grades 3 and 4 toxicities overall were significantly (P = 0 · 018) more common in the former group. The disease-free interval of the carboplatin and metronomic group was 192 days, which was not significantly different (P = 0 · 916) to the 182 days for the carboplatin alternating with doxorubicin and metronomic group. The median survival times of the two groups were 217 and 189 days, respectively. Piroxicam and cyclophosphamide metronomic protocols can be safely administered in combination with maximal-tolerated dose chemotherapy protocols. A significantly higher frequency of toxicities was observed in dogs treated with the carboplatin and metronomic protocol.

BMC Veterinary Research, 2013

Background: Osteosarcoma (OSA) is the most common primary bone tumor of dogs and carries a poor p... more Background: Osteosarcoma (OSA) is the most common primary bone tumor of dogs and carries a poor prognosis despite aggressive treatment. An improved understanding of the biology of OSA is critically needed to allow for development of novel diagnostic, prognostic, and therapeutic tools. The surface-exposed proteome (SEP) of a cancerous cell includes a multifarious array of proteins critical to cellular processes such as proliferation, migration, adhesion, and inter-cellular communication. The specific aim of this study was to define a SEP profile of two validated canine OSA cell lines and a normal canine osteoblast cell line utilizing a biotinylation/streptavidin system to selectively label, purify, and identify surface-exposed proteins by mass spectrometry (MS) analysis. Additionally, we sought to validate a subset of our MS-based observations via quantitative real-time PCR, Western blot and semiquantitative immunocytochemistry. Our hypothesis was that MS would detect differences in the SEP composition between the OSA and the normal osteoblast cells. Results: Shotgun MS identified 133 putative surface proteins when output from all samples were combined, with good consistency between biological replicates. Eleven of the MS-detected proteins underwent analysis of gene expression by PCR, all of which were actively transcribed, but varied in expression level. Western blot of whole cell lysates from all three cell lines was effective for Thrombospondin-1, CYR61 and CD44, and indicated that all three proteins were present in each cell line. Semi-quantitative immunofluorescence indicated that CD44 was expressed at much higher levels on the surface of the OSA than the normal osteoblast cell lines. Conclusions: The results of the present study identified numerous differences, and similarities, in the SEP of canine OSA cell lines and normal canine osteoblasts. The PCR, Western blot, and immunocytochemistry results, for the subset of proteins evaluated, were generally supportive of the mass spectrometry data. These methods may be applied to other cell lines, or other biological materials, to highlight unique and previously unrecognized differences between samples. While this study yielded data that may prove useful for OSA researchers and clinicians, further refinements of the described techniques are expected to yield greater accuracy and produce a more thorough SEP analysis.

BMC Veterinary Research, 2013

A critical step in developing therapeutics for oxidative stress-related pathologies is the abilit... more A critical step in developing therapeutics for oxidative stress-related pathologies is the ability to determine which specific modified protein species are innocuous by-products of pathology and which are causative agents. To achieve this goal, technologies are needed that can identify, characterize and quantify oxidative post translational modifications (oxPTMs). Nanobodies (Nbs) represent exquisite tools for intracellular tracking of molecules due to their small size, stability and engineerability. Here, we demonstrate that it is possible to develop a selective Nb against an oxPTM protein, with the key advance being the use of genetic code expansion (GCE) to provide an efficient source of the large quantities of high-quality, homogenous and site-specific oxPTM-containing protein needed for the Nb selection process. In this proof-of-concept study, we produce a Nb selective for a 3-nitrotyrosine (nitroTyr) modified form of the 14-3-3 signaling protein with a lesser recognition of ni...

16 Transitional cell carcinoma (TCC), the most common cancer of the urinary bladder in 17 dogs, i... more 16 Transitional cell carcinoma (TCC), the most common cancer of the urinary bladder in 17 dogs, is usually diagnosed at an advanced disease stage with limited response to chemotherapy. 18 Commercial screening tests lack specificity and current diagnostic procedures are invasive. A 19 proof of concept pilot project for analyzing the canine urinary proteome as a non-invasive 20 diagnostic tool for TCC identification was conducted. Urine was collected from 12 dogs in three 21 cohorts (healthy, urinary tract infection, TCC) and analyzed using liquid chromatography tandem 22 mass spectrometry. The presence of four proteins (macrophage capping protein, peroxiredoxin 5, 23 heterogeneous nuclear ribonucleoproteins A2/B, and apolipoprotein A1) was confirmed via 24 immunoblot. Of the total 379 proteins identified, 96 were unique to the TCC group. A statistical 25 model, designed to evaluate the accuracy of this multiplex biomarker approach for diagnosis of 26 TCC, predicted the presence of di...

Journal of Veterinary Internal Medicine, 2020

Background Lymphoma (LSA) is a common malignancy in dogs. Epigenetic changes are linked to LSA pa... more Background Lymphoma (LSA) is a common malignancy in dogs. Epigenetic changes are linked to LSA pathogenesis and poor prognosis in humans, and LSA pathogenesis in dogs. Sulforaphane (SFN), an epigenetic‐targeting compound, has recently gained interest in relation to cancer prevention and therapy. Objective Examine the impact of oral supplementation with SFN on the lymph node proteome of dogs with multicentric LSA. Animals Seven client‐owned dogs with multicentric LSA. Methods Prospective, nonrandomized, noncontrolled study in treatment‐naïve dogs with intermediate or large cell multicentric LSA. Lymph node cell aspirates were obtained before and after 7 days of oral supplementation with SFN, and analyzed via label‐free mass spectrometry, immunoblots, and Gene Set Enrichment Analysis. Results There was no clinical response and no adverse events attributed to SFN. For individual dogs, the expression of up to 650 proteins changed by at least 2‐fold (range, 2‐100) after supplementation w...

... The goal of this study was to identify prognostic indicators for canine hemangiosarcoma patie... more ... The goal of this study was to identify prognostic indicators for canine hemangiosarcoma patients treated with DAV. The focuses of this study were the c-kit receptor and the PI3k signaling pathway that have been associated with the pathology of many tumors. ...

Fasting has been shown to decrease chemotherapy‐associated adverse events (AEs), in part through ... more Fasting has been shown to decrease chemotherapy‐associated adverse events (AEs), in part through insulin‐like growth factor (IGF‐1) reduction, and may induce a protective effect on normal cells during chemotherapy treatment in mice and people. The purpose of this study was to evaluate the effect of fasting on constitutional, bone marrow and gastrointestinal (GI) AEs, and serum glucose, IGF‐1 and insulin levels in dogs receiving vincristine. The study was a prospective, crossover clinical trial in tumour‐bearing dogs. Dogs were randomized to be fasted for 24 to 28 hours prior to and 6 hours following their first or second vincristine treatment, and fed normally for the alternate dose. A significant reduction in nausea, anorexia, lethargy and serum insulin was observed when dogs were fasted; however, no significant differences were found in other GI symptoms, neutrophil count, serum glucose or IGF‐1. Fasting prior to vincristine therapy is a safe and effective treatment modality that ...

Cancer Cell International

Background Osteosarcoma patients often experience poor outcomes despite chemotherapy treatment, l... more Background Osteosarcoma patients often experience poor outcomes despite chemotherapy treatment, likely due in part to various mechanisms of tumor cell innate and/or acquired drug resistance. Exosomes, microvesicles secreted by cells, have been shown to play a role in drug resistance, but a comprehensive protein signature relating to osteosarcoma carboplatin resistance has not been fully characterized. Methods In this study, cell lysates and exosomes from two derivatives (HMPOS-2.5R and HMPOS-10R) of the HMPOS osteosarcoma cell line generated by repeated carboplatin treatment and recovery, were characterized proteomically by mass spectrometry. Protein cargos of circulating serum exosomes from dogs with naturally occurring osteosarcoma, were also assessed by mass spectrometry, to identify biomarkers that discriminate between good and poor responders to carboplatin therapy. Results Both cell lysates and exosomes exhibited distinct protein signatures related to drug resistance. Furtherm...

American Journal of Veterinary Research

Journal of Feline Medicine and Surgery Open Reports

Case summary A case of nasal adenocarcinoma as a suspected secondary malignant neoplasm following... more Case summary A case of nasal adenocarcinoma as a suspected secondary malignant neoplasm following definitive radiation therapy and multiagent chemotherapy for nasal lymphoma is described. An 11-year-old spayed female domestic shorthair cat was presented for a 3-week history of progressive facial swelling located over the nasal planum and extending to the medial canthus of the right eye. The cat was previously diagnosed with nasal lymphoma and treated with chemotherapy and definitive radiation 2.5 years prior. Although a definitive diagnosis could not be obtained via cytology, recurrent lymphoma was suspected based on the cat’s history and recurrent clinical signs. A lymphoma-directed chemotherapy protocol was attempted, but no clinical response was achieved. The cat was euthanased owing to progressive clinical signs and a diagnosis of nasal adenocarcinoma was made on necropsy examination. Both the original diagnosis of nasal lymphoma and the secondary diagnosis of nasal adenocarcino...

Cell Stress and Chaperones, 2022

Table S1. Nucleotide sequences of specific PCR primers. (PDF 336 kb)

Figure S1. Comparison of nucleotide sequences of kiss1 (A) and amino acid sequences of Kiss-1 (B)... more Figure S1. Comparison of nucleotide sequences of kiss1 (A) and amino acid sequences of Kiss-1 (B) in the dog, human, and mouse. (PDF 462 kb)

Analytical and Bioanalytical Chemistry, 2021

Separation of blood plasma or serum from blood is essential for accurate analysis. Conventional b... more Separation of blood plasma or serum from blood is essential for accurate analysis. Conventional blood separation requires instrumentation, reagents, and large sample volumes, limiting this process to laboratory environments with trained personnel. Full implementation of effective blood separation and analysis on microliter sample volumes for point of care (POC) diagnostics has proven extremely challenging resulting in a growing market demand, with common challenges such as expensive device fabrication processes or devices being comprised of materials which are not easily disposable. We developed a membrane-based wicking microfluidic device which is made using a simple fabrication process. This device uses a unique 3D flow channel geometry, fabricated in a polycaprolactone-filled glass microfiber membrane, to efficiently separate microliter sample volumes of blood. Colorimetric assay chemistries were integrated to demonstrate utility of these devices in POC diagnostics. The devices are capable of separating both fresh and anticoagulant-treated blood at microscale sample volumes (<15.0 μL). Modifications to the base device are also reported herein which increased sample volume capacity and separation efficiency. Integrated colorimetric assay enabled semi-quantitative detection of conjugated bilirubin in real blood samples (1.0–1.5 mg/dL). These blood separation devices, fabricated on polycaprolactone-filled glass microfiber, enabled effective blood plasma (anticoagulant-treated blood) and serum (fresh blood) separation with microscale sample volumes. Sample volume capacity and separation efficiency are customizable for specific applications and devices can be integrated with downstream assay chemistries to develop complete POC devices that offer blood separation and diagnostics at the same time on a single membrane.

Journal of the American Animal Hospital Association, 2009

A 9-year-old Bouvier des Flandres was presented with coughing, lethargy, chylous pleural effusion... more A 9-year-old Bouvier des Flandres was presented with coughing, lethargy, chylous pleural effusion, and a heart murmur. An echocardiogram revealed the presence of an intracardiac mass causing right ventricular outflow tract obstruction. The mass was successfully removed surgically, using total inflow occlusion. Histopathology and immunohistochemistry identified the tumor as an ectopic thyroid carcinoma. The dog was euthanized 11 months after diagnosis at the request of the owner because of nonresolving chylothorax.

Pediatric Blood & Cancer, 2013

Journal of Small Animal Practice, 2014

To evaluate the tolerability of a piroxicam and cyclophosphamide metronomic treatment protocol co... more To evaluate the tolerability of a piroxicam and cyclophosphamide metronomic treatment protocol combined with carboplatin alone or carboplatin and doxorubicin at maximal-tolerated doses. Retrospective study of 30 dogs diagnosed with osteosarcoma. All dogs underwent amputation and chemotherapy treatment with one of the two maximal-tolerated dose protocols. Metronomic chemotherapy was administered in conjunction with these protocols, and continued subsequently. The protocols included 0 · 3 mg/kg piroxicam and 10 to 12 mg/M(2) cyclophosphamide with 300 mg/M(2) carboplatin alone, or 300 mg/M(2) carboplatin alternating with 30 mg/M(2) doxorubicin. Fourteen dogs were treated with the carboplatin and metronomic protocol and 16 were treated with the carboplatin alternating with doxorubicin and metronomic protocol. Grades 3 and 4 toxicities overall were significantly (P = 0 · 018) more common in the former group. The disease-free interval of the carboplatin and metronomic group was 192 days, which was not significantly different (P = 0 · 916) to the 182 days for the carboplatin alternating with doxorubicin and metronomic group. The median survival times of the two groups were 217 and 189 days, respectively. Piroxicam and cyclophosphamide metronomic protocols can be safely administered in combination with maximal-tolerated dose chemotherapy protocols. A significantly higher frequency of toxicities was observed in dogs treated with the carboplatin and metronomic protocol.

BMC Veterinary Research, 2013

Background: Osteosarcoma (OSA) is the most common primary bone tumor of dogs and carries a poor p... more Background: Osteosarcoma (OSA) is the most common primary bone tumor of dogs and carries a poor prognosis despite aggressive treatment. An improved understanding of the biology of OSA is critically needed to allow for development of novel diagnostic, prognostic, and therapeutic tools. The surface-exposed proteome (SEP) of a cancerous cell includes a multifarious array of proteins critical to cellular processes such as proliferation, migration, adhesion, and inter-cellular communication. The specific aim of this study was to define a SEP profile of two validated canine OSA cell lines and a normal canine osteoblast cell line utilizing a biotinylation/streptavidin system to selectively label, purify, and identify surface-exposed proteins by mass spectrometry (MS) analysis. Additionally, we sought to validate a subset of our MS-based observations via quantitative real-time PCR, Western blot and semiquantitative immunocytochemistry. Our hypothesis was that MS would detect differences in the SEP composition between the OSA and the normal osteoblast cells. Results: Shotgun MS identified 133 putative surface proteins when output from all samples were combined, with good consistency between biological replicates. Eleven of the MS-detected proteins underwent analysis of gene expression by PCR, all of which were actively transcribed, but varied in expression level. Western blot of whole cell lysates from all three cell lines was effective for Thrombospondin-1, CYR61 and CD44, and indicated that all three proteins were present in each cell line. Semi-quantitative immunofluorescence indicated that CD44 was expressed at much higher levels on the surface of the OSA than the normal osteoblast cell lines. Conclusions: The results of the present study identified numerous differences, and similarities, in the SEP of canine OSA cell lines and normal canine osteoblasts. The PCR, Western blot, and immunocytochemistry results, for the subset of proteins evaluated, were generally supportive of the mass spectrometry data. These methods may be applied to other cell lines, or other biological materials, to highlight unique and previously unrecognized differences between samples. While this study yielded data that may prove useful for OSA researchers and clinicians, further refinements of the described techniques are expected to yield greater accuracy and produce a more thorough SEP analysis.

BMC Veterinary Research, 2013

A critical step in developing therapeutics for oxidative stress-related pathologies is the abilit... more A critical step in developing therapeutics for oxidative stress-related pathologies is the ability to determine which specific modified protein species are innocuous by-products of pathology and which are causative agents. To achieve this goal, technologies are needed that can identify, characterize and quantify oxidative post translational modifications (oxPTMs). Nanobodies (Nbs) represent exquisite tools for intracellular tracking of molecules due to their small size, stability and engineerability. Here, we demonstrate that it is possible to develop a selective Nb against an oxPTM protein, with the key advance being the use of genetic code expansion (GCE) to provide an efficient source of the large quantities of high-quality, homogenous and site-specific oxPTM-containing protein needed for the Nb selection process. In this proof-of-concept study, we produce a Nb selective for a 3-nitrotyrosine (nitroTyr) modified form of the 14-3-3 signaling protein with a lesser recognition of ni...

16 Transitional cell carcinoma (TCC), the most common cancer of the urinary bladder in 17 dogs, i... more 16 Transitional cell carcinoma (TCC), the most common cancer of the urinary bladder in 17 dogs, is usually diagnosed at an advanced disease stage with limited response to chemotherapy. 18 Commercial screening tests lack specificity and current diagnostic procedures are invasive. A 19 proof of concept pilot project for analyzing the canine urinary proteome as a non-invasive 20 diagnostic tool for TCC identification was conducted. Urine was collected from 12 dogs in three 21 cohorts (healthy, urinary tract infection, TCC) and analyzed using liquid chromatography tandem 22 mass spectrometry. The presence of four proteins (macrophage capping protein, peroxiredoxin 5, 23 heterogeneous nuclear ribonucleoproteins A2/B, and apolipoprotein A1) was confirmed via 24 immunoblot. Of the total 379 proteins identified, 96 were unique to the TCC group. A statistical 25 model, designed to evaluate the accuracy of this multiplex biomarker approach for diagnosis of 26 TCC, predicted the presence of di...

Journal of Veterinary Internal Medicine, 2020

Background Lymphoma (LSA) is a common malignancy in dogs. Epigenetic changes are linked to LSA pa... more Background Lymphoma (LSA) is a common malignancy in dogs. Epigenetic changes are linked to LSA pathogenesis and poor prognosis in humans, and LSA pathogenesis in dogs. Sulforaphane (SFN), an epigenetic‐targeting compound, has recently gained interest in relation to cancer prevention and therapy. Objective Examine the impact of oral supplementation with SFN on the lymph node proteome of dogs with multicentric LSA. Animals Seven client‐owned dogs with multicentric LSA. Methods Prospective, nonrandomized, noncontrolled study in treatment‐naïve dogs with intermediate or large cell multicentric LSA. Lymph node cell aspirates were obtained before and after 7 days of oral supplementation with SFN, and analyzed via label‐free mass spectrometry, immunoblots, and Gene Set Enrichment Analysis. Results There was no clinical response and no adverse events attributed to SFN. For individual dogs, the expression of up to 650 proteins changed by at least 2‐fold (range, 2‐100) after supplementation w...

... The goal of this study was to identify prognostic indicators for canine hemangiosarcoma patie... more ... The goal of this study was to identify prognostic indicators for canine hemangiosarcoma patients treated with DAV. The focuses of this study were the c-kit receptor and the PI3k signaling pathway that have been associated with the pathology of many tumors. ...

Fasting has been shown to decrease chemotherapy‐associated adverse events (AEs), in part through ... more Fasting has been shown to decrease chemotherapy‐associated adverse events (AEs), in part through insulin‐like growth factor (IGF‐1) reduction, and may induce a protective effect on normal cells during chemotherapy treatment in mice and people. The purpose of this study was to evaluate the effect of fasting on constitutional, bone marrow and gastrointestinal (GI) AEs, and serum glucose, IGF‐1 and insulin levels in dogs receiving vincristine. The study was a prospective, crossover clinical trial in tumour‐bearing dogs. Dogs were randomized to be fasted for 24 to 28 hours prior to and 6 hours following their first or second vincristine treatment, and fed normally for the alternate dose. A significant reduction in nausea, anorexia, lethargy and serum insulin was observed when dogs were fasted; however, no significant differences were found in other GI symptoms, neutrophil count, serum glucose or IGF‐1. Fasting prior to vincristine therapy is a safe and effective treatment modality that ...

Cancer Cell International

Background Osteosarcoma patients often experience poor outcomes despite chemotherapy treatment, l... more Background Osteosarcoma patients often experience poor outcomes despite chemotherapy treatment, likely due in part to various mechanisms of tumor cell innate and/or acquired drug resistance. Exosomes, microvesicles secreted by cells, have been shown to play a role in drug resistance, but a comprehensive protein signature relating to osteosarcoma carboplatin resistance has not been fully characterized. Methods In this study, cell lysates and exosomes from two derivatives (HMPOS-2.5R and HMPOS-10R) of the HMPOS osteosarcoma cell line generated by repeated carboplatin treatment and recovery, were characterized proteomically by mass spectrometry. Protein cargos of circulating serum exosomes from dogs with naturally occurring osteosarcoma, were also assessed by mass spectrometry, to identify biomarkers that discriminate between good and poor responders to carboplatin therapy. Results Both cell lysates and exosomes exhibited distinct protein signatures related to drug resistance. Furtherm...

American Journal of Veterinary Research

Journal of Feline Medicine and Surgery Open Reports

Case summary A case of nasal adenocarcinoma as a suspected secondary malignant neoplasm following... more Case summary A case of nasal adenocarcinoma as a suspected secondary malignant neoplasm following definitive radiation therapy and multiagent chemotherapy for nasal lymphoma is described. An 11-year-old spayed female domestic shorthair cat was presented for a 3-week history of progressive facial swelling located over the nasal planum and extending to the medial canthus of the right eye. The cat was previously diagnosed with nasal lymphoma and treated with chemotherapy and definitive radiation 2.5 years prior. Although a definitive diagnosis could not be obtained via cytology, recurrent lymphoma was suspected based on the cat’s history and recurrent clinical signs. A lymphoma-directed chemotherapy protocol was attempted, but no clinical response was achieved. The cat was euthanased owing to progressive clinical signs and a diagnosis of nasal adenocarcinoma was made on necropsy examination. Both the original diagnosis of nasal lymphoma and the secondary diagnosis of nasal adenocarcino...