Modeling and Molecular Dynamic Simulation of F(ab')2 Fragment of Nimotuzumab for Lung Cancer Diagnostics.

Lung cancer is one of the leading causes of cancer-related deaths in the world among both men and women. Several studies in the literature report that overexpression and mutation of the epidermal growth factor receptor (EGFR) are implicated in the pathogenesis of some lung cancers. Nimotuzumab is a humanized monoclonal antibody (mAb) that inhibits EGF binding because it binds to the extracellular domain of the EGFR. Nimotuzumab requires bivalent binding for stable attachment to cellular surface, which leads to nimotuzumab selectively binding to cells that express mAbs of moderate to high EGFR levels, and this could explain its low toxicity. This property has an advantage for development of nimotuzumab as a therapeutic and diagnostic agent. Monoclonal antibodies are large in size (150 kDa), thus penetrating slowly and residing in the blood for extended periods of time (from days to weeks); their use in imaging studies can result in low signal-to-background ratios and poor image quality. A reduction in the size of the immunoglobulin molecule has also been proposed as a means for increasing tumor penetration by mAbs. Nevertheless, it is known that the penetration of mAb into tumor cell is slow, due to its high molecular weight. Therefore, mAb is not very attractive to be used for imaging diagnostic purpose because of its kinetics and potential to elicit antibody response. The objective of this research was to study the homology modeling of a simpler functional molecule based on nimotuzumab, which consists of 2 antigen-binding fragments (Fab), namely, F(ab')2, using MODELER. The crystal structure of Fab of nimotuzumab from protein data bank was used as a template to construct the model of F(ab')2. Molecular dynamic simulation was performed to evaluate the stability of F(ab')2 and conformational changes of F(ab')2 in simulation. The result showed the dynamic behavior of antigen-binding site region of F(ab')2 throughout simulation. This result is expected to be useful in the further development of F(ab')2 fragment nimotuzumab as a lung cancer diagnostic.

Antigenic potential of a recombinant polyvalent DNA vaccine against pathogenic leptospiral infection.

Leptospirosis is a serious epidemic disease caused by pathogenic Leptospira species. The disease is endemic in most tropical and sub-tropical regions of the world. Currently, there is no effective polyvalent vaccine for prevention against most of the circulating serovars. Moreover, development of an efficient leptospiral vaccine capable of stimulating cross-protective immune responses against a wide range of serovars remains a daunting challenge. This, in part, is associated with the extensive diversity and variation of leptospiral serovars from region to region. In this study, a multi-epitope DNA vaccine encoding highly immunogenic epitopes from LipL32 and LipL41 was designed using in-silico approach. The DNA encoding antigenic epitopes was constructed from conserved pathogenic Leptospira genes (LipL32 and LipL41). Immunization of golden Syrian hamsters with the multi-epitope chimeric DNA vaccine resulted in the production of both agglutinating and neutralizing antibodies as evidence by MAT and in-vitro growth inhibition tests respectively. The antibodies produced reacted against eight different serovars and significantly reduced renal colonization following in vivo challenge. The vaccine was also able to significantly reduce renal colonization which is a very important factor responsible for persistence of leptospires among susceptible and reservoir animal hosts. In conclusion, the leptospiral multi-epitope chimeric DNA vaccine can serve as a potentially effective and safe vaccine against infection with different pathogenic leptospiral serovars.

Computational Study of Imidazolylporphyrin Derivatives as a Radiopharmaceutical Ligand for Melanoma.

BACKGROUND: Melanoma is the most aggressive type of skin cancer. Metastatic melanoma is extremely difficult to treat with current therapy methods such as surgery. On the other hand, it is a good opportunity to develop a radiopharmaceutical using a radionuclide such as Technetium (Tc) for diagnostic and Rhenium (Re) for therapeutic purposes. T3,4BCPP has been be used as a radioimaging agent for melanoma cancers experimentally. The aim of the present research was to design new imidazolylporphyrin derivatives with better selectivity and higher affinity than those of T3,4BCPP by molecular modeling. METHODS: Eight types of Re- and Tc-labeled imidazolylporphyrins were docked to Fibroblast Growth Factor Receptor 1 (FGFR1, PDB ID: 5AM6) using AutoDock 4.2. FGFR1 was simulated by Molecular Dynamic (MD) simulation for 30 ns using NAMD 2.10 at 37 °C. The obtained conformations were then applied in a molecular docking simulation. Dovitinib (natural ligand of FGFR1), Re- and Tc-T3, 4BCPP were used as references. RESULTS: The MD simulation resulted in an RMSD of 3.8 Å. From all the studied imidazolylporphyrin derivatives, Tc-cD3, 4BCPMIP and Re-cD3, 4BCPIP had the best docking parameter. Tc-cD3, 4BCPMIP had a free binding energy of -4.06 kcal/mol, while that of Re-cD3, 4BCPIP was -4.35 kcal/mol. CONCLUSION: It is concluded that cD3,4BCPMIP and cD3,4BCPIP are two potential candidate ligands for a melanoma radiopharmaceutical kit.

In Silico Study, Synthesis, and Cytotoxic Activities of Porphyrin Derivatives.

Five known porphyrins, 5,10,15,20-tetrakis(p-tolyl)porphyrin (TTP), 5,10,15,20-tetrakis(p-bromophenyl)porphyrin (TBrPP), 5,10,15,20-tetrakis(p-aminophenyl)porphyrin (TAPP), 5,10,15-tris(tolyl)-20-mono(p-nitrophenyl)porphyrin (TrTMNP), 5,10,15-tris(tolyl)-20-mono(p-aminophenyl)porphyrin (TrTMAP), and three novel porphyrin derivatives, 5,15-di-[bis(3,4-ethylcarboxymethylenoxy)phenyl]-10,20-di(p-tolyl)porphyrin (DBECPDTP), 5,10-di-[bis(3,4-ethylcarboxymethylenoxy)phenyl]-15,20-di-(methylpyrazole-4-yl)porphyrin (cDBECPDPzP), 5,15-di-[bis(3,4-ethylcarboxymethylenoxy)phenyl]-10,20-di-(methylpyrazole-4-yl)porphyrin (DBECPDPzP), were used to study their interaction with protein targets (in silico study), and were synthesized. Their cytotoxic activities against cancer cell lines were tested using 3-(4,5-dimetiltiazol-2-il)-2,5-difeniltetrazolium bromide (MTT) assay. The interaction of porphyrin derivatives with carbonic anhydrase IX (CAIX) and REV-ERBß proteins were studied by molecular docking and molecular dynamic simulation. In silico study results reveal that DBECPDPzP and TrTMNP showed the highest binding interaction with REV- ERBß and CAIX, respectively, and both complexes of DBECPDPzP-REV-ERBß and TrTMNP-CAIX showed good and comparable stability during molecular dynamic simulation. The studied porphyrins have selective growth inhibition activities against tested cancer cells and are categorized as marginally active compounds based on their IC50.

Major epidemiological factors associated with leptospirosis in Malaysia.

INTRODUCTION: Leptospirosis is a zoonotic disease caused by a diverse pathogenic leptospira species and serovars. The disease is transmitted directly following contact with infected urine and other body fluids or indirectly after contact with water or soil contaminated with infected urine. OBJECTIVES: While a wide range of domestic and wild animals are known to be reservoirs of the disease, occupation, international travel and recreation are beginning to assume a center stage in the transmission of the disease. The objective of this study is to review available literatures to determine the extent to which these aforementioned risk factors aid the transmission, increase incidence and outbreak of leptospirosis in Malaysia. STUDY DESIGN: The review was conducted based on prevalence, incidence, and outbreak cases of leptospirosis among human and susceptible animals predisposed to several of the risk factors identified in Malaysia. METHODS: Literature searchers and reviews were conducted based on articles published in citation index journals, Malaysian ministry of health reports, periodicals as well as reliable newspapers articles and online media platforms. In each case, the newspapers and online media reports were supported by press briefings by officials of the ministry of health and other agencies responsible. RESULTS: The disease is endemic in Malaysia, and this was attributed to the large number of reservoir animals, suitable humid and moist environment for proliferation as well as abundant forest resources. Over 30 different serovars have been detected in Malaysia in different domestic and wild animal species. This, in addition to the frequency of flooding which has increased in recent years, and has helped increase the risk of human exposure. Occupation, recreation, flooding and rodent population were all identified as an important source and cause of the disease within the study population. CONCLUSION: There is an urgent need for the government and other stakeholders to intensify efforts to control the spread of the disease, especially as it greatly affect human health and the tourism industry which is an important component of the Malaysian economy. The risk of infection can be minimized by creating awareness on the source and mode of transmission of the disease, including the use of protective clothing and avoiding swimming in contaminated waters. Moreover, improved diagnostics can also help reduce the suffering and mortalities that follow infection after exposure to infection source.

Retrospective Study of Leptospirosis in Malaysia.

Leptospirosis is a bacterial disease transmitted to humans and animals by direct or indirect contact with urine or body fluids from infected animals especially rodents. Infection can be associated with wide clinical spectrum varying from asymptomatic to severe multi-organ syndrome with life-threatening consequences. We conducted a review of published studies on incidences, case reports, sero-epidemiological surveys from year 2000 to 2015 using different electronic data bases. Our study revealed that majority of the studies were conducted in Peninsular Malaysia and predominantly among high-risk human groups. Most of the studies on domestic animals were conducted in the 1980s; hence, the current status of leptospirosis among domestic animal population remains largely unknown. There tend to be a sharp rise in incidence rate among human population in the year 2014 which was attributed to flooding and heavy rainfall experienced as well as recreational activities. Several gaps in epidemiological knowledge were also disclosed.

Evidence and potential risk factors of tuberculosis among captive Asian elephants and wildlife staff in Peninsular Malaysia.

Elephant tuberculosis (TB) caused by Mycobacterium tuberculosis is an important re-emerging zoonosis with considerable conservation and public health risk. We conducted prospective cohort and cross-sectional studies in elephants and wildlife staff respectively in order to identify potential risk factors associated with TB in captive Asian elephants and their handlers in Peninsular Malaysia. Sixty elephants in six different facilities were screened for TB longitudinally using the ElephantTB STAT-PAK and DPP VetTB assays from February 2012 to May 2014, and 149 wildlife staff were examined for tuberculosis infection using the QuantiFERON-TB Gold In-tube (QFT) assay from January to April, 2012. Information on potential risk factors associated with infection in both elephants and staff were collected using questionnaires and facility records. The overall seroprevalence of TB amongst the elephants was 23.3% (95% CI: 13.8-36.3) and the risk of seroconversion was significantly higher among elephants with assigned mahouts [p=0.022, OR=4.9 (95% CI: 1.3-18.2)]. The percentage of QFT responders among wildlife staff was 24.8% (95% CI: 18.3-32.7) and the risk of infection was observed to be significantly associated with being a zoo employee [p=0.018, OR=2.7 (95% CI: 1.2-6.3)] or elephant handler [p=0.035, OR=4.1 (95% CI: 1.1-15.5)]. These findings revealed a potential risk of TB infection in captive elephants and handlers in Malaysia, and emphasize the need for TB screening of newly acquired elephants, isolating sero-positive elephants and performing further diagnostic tests to determine their infection status, and screening elephant handlers for TB, pre- and post-employment.

Laws, Regulations, Guidelines and Standards for Animal Care and Use for Scientific Purposes in the Countries of Singapore, Thailand, Indonesia, Malaysia, and India.

The laws, regulations, guidelines, and standards on animal care and use for scientific purposes in the countries of Singapore, Thailand, Indonesia, and Malaysia, and India are described in this manuscript. For each of these five countries, a brief introduction is provided on the history of how the need for animal welfare in research, education, training, and testing came to being. This is followed by some background information leading to the current status of regulations and guidelines in each of the five countries. There is also a description of the responsibilities and functions of institutional animal welfare and ethics oversight bodies, enforcement agencies, penalties, and organizations supporting the industry. Finally, a conclusion with insights into the future of laboratory animal welfare and science in each of these five countries in Asia is provided.

Production and characterization of a polyclonal antibody of anti-rLipL21-IgG against leptospira for early detection of acute leptospirosis.

Leptospirosis is one of the zoonotic diseases in animals and humans throughout the world. LipL21 is one of the important surface-exposed lipoproteins in leptospires and the most effective cross protective immunogenic antigen. It is widely considered as a diagnostic marker for leptospirosis. In this study, we evaluated the serodiagnostic potential of LipL21 protein of Leptospira interrogans serovar Pomona. We have successfully amplified, cloned, and expressed LipL21 in E. coli and evaluated its specificity by immunoblotting. Purified recombinant LipL21 (rLipL21) was inoculated into rabbits for the production of polyclonal antibody. Characterization of the purified IgG antibody against rLipL21 was performed by cross reactivity assay. Only sera from leptospirosis patients and rabbit hyperimmune sera recognized rLipL21 while the nonleptospirosis control sera showed no reaction in immunoblotting. We confirmed that anti-rLipL21-IgG antibody cross reacted with and detected only pathogenic leptospiral species and it did not react with nonpathogenic leptospires and other bacterial species. Results observed showed that anti-rLipL21-IgG antibody has high specificity and sensitivity to leptospires. The findings indicated that the antibody could be used in a diagnostic assay for detection of leptospires or their proteins in the early phase of infection.

Molecular characterization of Pasteurella multocida isolates from rabbits.

Forty isolates of Pasteurella multocida from healthy (17 isolates) and diseased (23 isolates) rabbits were assayed for the presence of plasmids in seeking to determine whether any correlation exists between the presence of plasmids and health status, sensitivity to antimicrobial agents, capsular and somatic type, and the anatomic site of isolation. Six isolates were found harboring plasmids. A similar ladder pattern ranging from 18 to 3 megadalton (Mda) were found in three isolates recovered from diseased rabbits. One band of molecular weight 6.6 Mda was shared by four of five (4/5) isolates from the diseased rabbits. No correlation was found between the presence of the common plasmids and serotype, resistance to antimicrobial agents, and anatomic sites from which the bacteria were cultured. Random amplification polymorphic DNA was applied to subtype all the isolates of P. multocida. Two single primers were tested for their abilities to generate individual fingerprints by using PCR. Primer 1 grouped the isolates into 7 profiles, and primer 2 grouped them into 15. Random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) results show the presence of a wide heterogeneity within P. multocida isolates. Therefore RAPD-PCR is an efficient technique to detect the DNA polymorphism and could be used to discriminate P. multocida of rabbit isolates together with serologic typing.