Genetically Engineered Hepatitis C Virus-like Particles (HCV-LPs) Tagged with SP94 Peptide to Acquire Selectivity to Liver Cancer Cells via Grp78.

Targeted cancer therapy is a challenging area that includes multiple chemical and biological vehicles. Virus-like particles (VLPs) combine safety and efficacy in their roles as potential vaccines and drug delivery vehicles. In this study, we propose a novel drug delivery system based on HCV-LPs engineered with SP94 and RGD peptides mediated by a specific molecular chaperone (Grp78) associated with cancer drug resistance. The PCR primers were designed for engineering two constructs, SP94-EGFP-CORE-HIS and RGD-EGFP-CORE-HIS, by sequential PCR reactions. The two fragments were cloned into pFastBac Dual under the polyhedrin promoter and then used to produce two recombinant baculoviruses (AcSP94 and AcRGD). The VLP's expression was optimized by recombinant virus infection with different MOIs, ranging from 1 to 20 MOI. Recombinant VLP2 were purified by Ni-NTA and their sizes and shapes were confirmed with TEM. They were incubated with different types of cells prior to examination using the fluorescence microscope to test the binding specificity. The effect of the overexpression of the Grp78 on the binding affinity of the engineered VLPs was tested in HepG2 and HeLa cells. The protocol optimization revealed that MOI 10 produced the highest fluorescence intensities after 72 h for the two recombinant proteins (SP94-core and RGD-core). Moreover, the binding assay tested on different types of mammalian cells (HeLa, HEK-293T, and HepG2 cells) showed green fluorescence on the periphery of all tested cell lines when using the RGD-core protein; while, the SP94-core protein showed green fluorescence only with the liver cancer cells, HepG2 and HuH7. Overexpression of Grp78 in HepG2 and HeLa cells enhanced the binding efficiency of the engineered VLPs. We confirmed that the SP94 peptide can be specifically used to target liver cancer cells, while the RGD peptide is sufficiently functional for most types of cancer cells. The overexpression of the Grp78 improved the binding capacity of both SP94 and RGD peptides. It is worth noting that the SP94 peptide can function properly as a recombinant peptide, and not only as a chemically conjugated peptide, as heretofore commonly used.

Lack of Annexin A6 Exacerbates Liver Dysfunction and Reduces Lifespan of Niemann-Pick Type C Protein-Deficient Mice.

Niemann-Pick type C (NPC) disease is a lysosomal storage disorder characterized by cholesterol accumulation caused by loss-of-function mutations in the Npc1 gene. NPC disease primarily affects the brain, causing neuronal damage and affecting motor coordination. In addition, considerable liver malfunction in NPC disease is common. Recently, we found that the depletion of annexin A6 (ANXA6), which is most abundant in the liver and involved in cholesterol transport, ameliorated cholesterol accumulation in Npc1 mutant cells. To evaluate the potential contribution of ANXA6 in the progression of NPC disease, double-knockout mice (Npc1-/-/Anxa6-/-) were generated and examined for lifespan, neurologic and hepatic functions, as well as liver histology and ultrastructure. Interestingly, lack of ANXA6 in NPC1-deficient animals did not prevent the cerebellar degeneration phenotype, but further deteriorated their compromised hepatic functions and reduced their lifespan. Moreover, livers of Npc1-/-/Anxa6-/- mice contained a significantly elevated number of foam cells congesting the sinusoidal space, a feature commonly associated with inflammation. We hypothesize that ANXA6 deficiency in Npc1-/- mice not only does not reverse neurologic and motor dysfunction, but further worsens overall liver function, exacerbating hepatic failure in NPC disease.

Linking Late Endosomal Cholesterol with Cancer Progression and Anticancer Drug Resistance.

Cancer cells undergo drastic metabolic adaptions to cover increased bioenergetic needs, contributing to resistance to therapies. This includes a higher demand for cholesterol, which often coincides with elevated cholesterol uptake from low-density lipoproteins (LDL) and overexpression of the LDL receptor in many cancers. This implies the need for cancer cells to accommodate an increased delivery of LDL along the endocytic pathway to late endosomes/lysosomes (LE/Lys), providing a rapid and effective distribution of LDL-derived cholesterol from LE/Lys to other organelles for cholesterol to foster cancer growth and spread. LDL-cholesterol exported from LE/Lys is facilitated by Niemann-Pick Type C1/2 (NPC1/2) proteins, members of the steroidogenic acute regulatory-related lipid transfer domain (StARD) and oxysterol-binding protein (OSBP) families. In addition, lysosomal membrane proteins, small Rab GTPases as well as scaffolding proteins, including annexin A6 (AnxA6), contribute to regulating cholesterol egress from LE/Lys. Here, we summarize current knowledge that links upregulated activity and expression of cholesterol transporters and related proteins in LE/Lys with cancer growth, progression and treatment outcomes. Several mechanisms on how cellular distribution of LDL-derived cholesterol from LE/Lys influences cancer cell behavior are reviewed, some of those providing opportunities for treatment strategies to reduce cancer progression and anticancer drug resistance.

Annexin A6 Is Critical to Maintain Glucose Homeostasis and Survival During Liver Regeneration in Mice.

BACKGROUND AND AIMS: Liver regeneration requires the organized and sequential activation of events that lead to restoration of hepatic mass. During this process, other vital liver functions need to be preserved, such as maintenance of blood glucose homeostasis, balancing the degradation of hepatic glycogen stores, and gluconeogenesis (GNG). Under metabolic stress, alanine is the main hepatic gluconeogenic substrate, and its availability is the rate-limiting step in this pathway. Na+ -coupled neutral amino acid transporters (SNATs) 2 and 4 are believed to facilitate hepatic alanine uptake. In previous studies, we demonstrated that a member of the Ca2+ -dependent phospholipid binding annexins, Annexin A6 (AnxA6), regulates membrane trafficking along endo- and exocytic pathways. Yet, although AnxA6 is abundantly expressed in the liver, its function in hepatic physiology remains unknown. In this study, we investigated the potential contribution of AnxA6 in liver regeneration. APPROACH AND RESULTS: Utilizing AnxA6 knockout mice (AnxA6-/- ), we challenged liver function after partial hepatectomy (PHx), inducing acute proliferative and metabolic stress. Biochemical and immunofluorescent approaches were used to dissect AnxA6-/- mice liver proliferation and energetic metabolism. Most strikingly, AnxA6-/- mice exhibited low survival after PHx. This was associated with an irreversible and progressive drop of blood glucose levels. Whereas exogenous glucose administration or restoration of hepatic AnxA6 expression rescued AnxA6-/- mice survival after PHx, the sustained hypoglycemia in partially hepatectomized AnxA6-/- mice was the consequence of an impaired alanine-dependent GNG in AnxA6-/- hepatocytes. Mechanistically, cytoplasmic SNAT4 failed to recycle to the sinusoidal plasma membrane of AnxA6-/- hepatocytes 48 hours after PHx, impairing alanine uptake and, consequently, glucose production. CONCLUSIONS: We conclude that the lack of AnxA6 compromises alanine-dependent GNG and liver regeneration in mice.

Annexin Animal Models-From Fundamental Principles to Translational Research.

Routine manipulation of the mouse genome has become a landmark in biomedical research. Traits that are only associated with advanced developmental stages can now be investigated within a living organism, and the in vivo analysis of corresponding phenotypes and functions advances the translation into the clinical setting. The annexins, a family of closely related calcium (Ca2+)- and lipid-binding proteins, are found at various intra- and extracellular locations, and interact with a broad range of membrane lipids and proteins. Their impacts on cellular functions has been extensively assessed in vitro, yet annexin-deficient mouse models generally develop normally and do not display obvious phenotypes. Only in recent years, studies examining genetically modified annexin mouse models which were exposed to stress conditions mimicking human disease often revealed striking phenotypes. This review is the first comprehensive overview of annexin-related research using animal models and their exciting future use for relevant issues in biology and experimental medicine.

Combined open reduction and Dega transiliac osteotomy for developmental dysplasia of the hip in walking children.

The iliac osteotomy described by Dega in Poland, in 1969, is an acetabuloplasty that changes the acetabular configuration and its inclination. The aim of this work is to analyze a group of patients with DDH treated by combined open reduction and Dega transiliac osteotomy ,to evaluate the results and determine the advantages and disadvantages, as well as, assess the factors affecting the final outcome of such procedure. A prospective study was conducted during the period, from November 2010 to October 2014, on 39 hips, in 29 children, with neglected DDH after walking age, either diagnosed late or after failure to respond to previous non operative treatment. The mean age at the time of surgery was 27.6 months ranging from 18 to 48 months. All hips were followed up clinically and radiologically for a mean period of 33.6 months (range from 18 to 48 months). No patient was lost to follow up. At the end of follow up, satisfactory final clinical results were obtained in 34 hips (87.2%) and unsatisfactory in 5 (12.8%) according to McKay's criteria. Radiologically, satisfactory results were obtained in 32 hips (82.1%) and unsatisfactory in seven (17.9%), according to Severin's criteria. In conclusion, the results of our series show open reduction combined with Dega transiliac osteotomy to be a safe and efficient method for the surgical treatment of DDH in selected patients, and can easily and safely be combined with associated procedures for single stage correction of acetabular dysplasia.

Assessment of psychological alarms and coping strategies of medical students with irritable bowel syndrome at Zagazig University: A cross-sectional study.

BACKGROUND: Irritable bowel syndrome (IBS) is one of the most prevalent functional gastrointestinal disorders. Medical students tend to report a higher prevalence of IBS since they are under constant stress. Many psychological difficulties are associated with IBS. To cope with IBS, individuals use various strategies which can impact the intensification or alleviation of IBS symptoms. The objective of this study was to assess the prevalence of IBS in medical students as well as psychological alarms and coping strategie employed by IBS sufferers. MATERIALS AND METHODS: We conducted a cross-sectional study from December 2022 to February 2023. Study participants were first to fifth year medical school students at Zagazig University, Egypt. Data were collected using a structured questionnaire comprising four sections: sociodemographic and clinical data; Rome IV criteria for the diagnosis of IBS; the alarm questionnaire for functional gastrointestinal disorders; and the Coping Strategies Questionnaire (CSQ24). Chi-square test or Fischer's exact test, as appropriate, were used to test for association. Binary logistic regression with a backward stepwise method was used to determine significant risk factors of negative coping with IBS. RESULTS: Of the studied 221 medical students, 38% had IBS. A statistically significant association was observed between IBS and the feeling of tension, anxiety, nervousness, depression, and frustration in the previous week, severe pain in the past 4 weeks, and the feeling that the bad situation would not get any better. Most of the students in the IBS group coped positively with stress, while 19.0% were negative in coping. Pain affecting the daily activities and the feelings of depression and frustration to the point of self-harm or suicide were the most significant correlates of IBS group's inability to cope. CONCLUSION: The prevalence of IBS in medical students at Zagazig University was 38%. We recommend psychological intervention and stress management programs to help medical students cope with IBS.

Assessment of groundwater quality in arid regions utilizing principal component analysis, GIS, and machine learning techniques.

Assessing water quality in arid regions is vital due to scarce resources, impacting health and sustainable management.This study examines groundwater quality in Assuit Governorate, Egypt, using Principal Component Analysis, GIS, and Machine Learning Techniques. Data from 217 wells across 12 parameters were analyzed, including TDS, EC, Cl-, Fe++, Ca++, Mg++, Na+, SO4--, Mn++, HCO3-, K+, and pH. The Water Quality Index (WQI) was calculated, and ArcGIS mapped its spatial distribution. Machine learning algorithms, including Ridge Regression, XGBoost, Decision Tree, Random Forest, and K-Nearest Neighbors, were used for predictive analysis. Higher concentrations of Na, K, Ca, Mg, Mn, and Fe were correlated with industrial and densely populated areas. Most samples exhibited excellent or good quality, with a small percentage unsuitable for consumption. Ridge Regression showed the lowest MAPE rates (0.22 % training, 0.26 % in testing). This research highlights the importance of advanced machine learning for sustainable groundwater management in arid regions. Thus, our results could provide valuable assistance to both national and local authorities involved in water management decisions, particularly for water resource managers and decision-makers. This information can aid in the development of regulations aimed at safeguarding and sustainably managing groundwater resources, which are essential for the overall prosperity of the country.

Forecasting of flash flood susceptibility mapping using random forest regression model and geographic information systems.

Flash floods, rapid and devastating inundations of water, are increasingly linked to the intensifying effects of climate change, posing significant challenges for both vulnerable communities and sustainable environmental management. The primary goal of this research is to investigate and predict a Flood Susceptibility Map (FSM) for the Ibaraki prefecture in Japan. This research utilizes a Random Forest (RF) regression model and GIS, incorporating 11 environmental variables (involving elevation, slope, aspect, distance to stream, distance to river, distance to road, land cover, topographic wetness index, stream power index, and plan and profile curvature), alongside a dataset comprising 224 instances of flooded and non-flooded locations. The data was randomly classified into a 70 % training set for model development, with the remaining 30 % used for model validation through Receiver Operating Characteristics (ROC) curve analysis. The resulting map indicated that approximately two-thirds of the prefecture as exhibiting low to very low flood susceptibility, while approximately one-fifth of the region is categorized as high to very high flood susceptibility. Furthermore, the RF model achieved a noteworthy validation with an area under the ROC curve of 99.56 %. Ultimately, this FSM serves as a crucial tool for policymakers in guiding appropriate spatial planning and flood mitigation strategies.

Baculovirus displaying SARS-CoV-2 spike RBD promotes neutralizing antibody production in a mouse model.

BACKGROUND: There is always a need for a safe and efficient vaccine platform, especially when facing a pandemic such as COVID-19. Most of the SARS-CoV-2-based vaccines are based on the full spike protein, which is presented as a trimerized protein, and many viral vector vaccines express the spike protein into the host cells and do not display it on virus surfaces. However, the spike receptor-binding domain (RBD)-based vaccines are efficient and are currently under investigation and clinical trials. METHODOLOGY: In this study, we are testing the efficacy of the RBD displayed on a baculovirus as a mean to formulate a safe and stable carrier to induce the immune system against SARS-CoV-2. Therefore, two pseudotyped baculoviruses were constructed to display the RBD, AcRBD-sfGFP-64, and AcRBD-sfGFP-V, using two different displaying strategies based on gp64 and VSV-G envelope glycoproteins, from Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and vesicular stomatitis virus (VSV), respectively. BALB/C mice were immunized with the pseudotyped baculoviruses in a dose-optimized manner. Dot blot and Western blot were used to screen and validate the polyclonal antibodies' specificity to the SARS-CoV-2 RBD. A plaque reduction neutralization test (PRNT) was used to measure the sera neutralization capacity against a SARS-CoV-2 wild-type isolate from Egypt. ELISA was used to quantify certain cytokines for the assessment of the immune response. RESULT: The outcome of our investigation showed that the monomeric RBD proteins were properly displayed on baculovirus and efficiently triggered the mouse immune system. The produced sera efficiently neutralized about 50% of SARS-CoV-2 in more than 100-fold serum dilution. The immunized mice showed a significant increase (p<0.01) in the levels of IL-2 and IFN-γ and a significant decrease (p<0.01) and (p<0.001) in the levels of IL-4 and IL-10, respectively, which suggest that AcRBD-sfGFP-64 and AcRBD-sfGFP-V induce Th1 cellular immune response. CONCLUSION: The produced recombinant viruses can induce the immune response without adjuvant, which needs dose optimization and further stability tests. Neutralizing antibodies were induced without affecting the health of immunized mice. Th1 response can be attainable through the system, which is of great benefit in SARS CoV-2 infection and the system can be tested for future applications including vaccine development and polyclonal antibody production.

Annexin A6 and NPC1 regulate LDL-inducible cell migration and distribution of focal adhesions.

Cholesterol is considered indispensable for cell motility, but how physiological cholesterol pools enable cells to move forward remains to be clarified. The majority of cells obtain cholesterol from the uptake of Low-Density lipoproteins (LDL) and here we demonstrate that LDL stimulates A431 squamous epithelial carcinoma and Chinese hamster ovary (CHO) cell migration and invasion. LDL also potentiated epidermal growth factor (EGF) -stimulated A431 cell migration as well as A431 invasion in 3-dimensional environments, using organotypic assays. Blocking cholesterol export from late endosomes (LE), using Niemann Pick Type C1 (NPC1) mutant cells, pharmacological NPC1 inhibition or overexpression of the annexin A6 (AnxA6) scaffold protein, compromised LDL-inducible migration and invasion. Nevertheless, NPC1 mutant cells established focal adhesions (FA) that contain activated focal adhesion kinase (pY397FAK, pY861FAK), vinculin and paxillin. Compared to controls, NPC1 mutants display increased FA numbers throughout the cell body, but lack LDL-inducible FA formation at cell edges. Strikingly, AnxA6 depletion in NPC1 mutant cells, which restores late endosomal cholesterol export in these cells, increases their cell motility and association of the cholesterol biosensor D4H with active FAK at cell edges, indicating that AnxA6-regulated transport routes contribute to cholesterol delivery to FA structures, thereby improving NPC1 mutant cell migratory behaviour.

Incidence and profile of acute intoxication among adult population in Najran, Saudi Arabia: A retrospective study.

Acute poisoning is considered one of the most important medical emergencies, resulting in severe morbidity and mortality, and is an economic burden on governments. This study aimed to determine the extent of acute adult intoxication among the population located in the Najran area, Saudi Arabia, over the last 3 years (from January 2017 to December 2019). The study is a hospital-based retrospective observational study. The data of all acutely intoxicated adult patients were collected from patients' files of King Khalid Hospital, the main hospital in the Najran area. In this study, the total number of intoxicated patients was 852. Patients were divided into three groups according to their age: 15-25 years, 26-35 years and >35 years. Accidental intoxication was predominant (64.6%), especially with therapeutic drugs (60.2%), predominantly acetaminophen and amphetamine, which intoxicated 24.5% and 23.4% of the patients, respectively. Moreover, this study showed that 10.6% of patients were intoxicated with overdoses of alcohol, mostly among patients aged over 35 years. Furthermore, the present study revealed that 23.9% of patients were intoxicated with household chemicals, especially Clorox bleach or Flash. Patients presented with a wide range of symptoms; some were even asymptomatic. Overall, patients' outcomes were good; mortalities were few (1.2%), and most fatalities were found in patients aged over 35 years (60%). The present study showed that pharmaceutical drugs constituted the most common causative agents in acute intoxication. Household chemicals, especially Clorox bleach, Flash and pesticides, are highly implicated in the acute toxicity problem. Drug abuse, especially amphetamine and alcohol, still represents a great threat facing people from the Najran region. It is crucial to deliver effective public health education programmes to increase community awareness about the predisposing risk factors of acute toxicity, whether as overdoses or suicide attempts.

De novo Portal Vein Thrombosis in Non-Cirrhotic Non-Alcoholic Fatty Liver Disease: A 9-Year Prospective Cohort Study.

Background and Aims: Approximately 30-40% of portal vein thrombosis (PVT) remains of unknown origin. The association between non-alcoholic fatty liver disease (NAFLD) and PVT is a matter of debate. This study aimed to investigate the association between PVT and NAFLD. Methods: We included 94 out of 105 consecutive NAFLD patients in this prospective cohort study in addition to 94 from the healthy control group. We evaluated biochemical, clinical, immunological, and histopathological parameters; waist circumference (WC); leptin; adiponectin; and leptin/adiponectin ratio (LAR) for all participants at baseline and every 3 years thereafter. We described the characteristics of participants at baseline and showed individual WC, LAR, and PVT characteristics. Potential parameters to predict PVT development within 9 years were determined. Results: PVT developed in eight (8.5%) patients, mainly in the portal trunk. Univariate analysis showed three PVT-associated factors: diabetes mellitus (P = 0.013), WC (P < 0.001), and LAR (P = 0.002). After adjusting multiple confounding variables, the multivariate model showed that the only significant variables were WC and LAR. By applying the receiver operating characteristic curve, WC had 98.8% specificity, 87.5% sensitivity, and 0.894 area under the curve (AUC) for prediction of PVT (P < 0.001) at cutoff values of > 105 cm. In comparison, LAR had 60.5% specificity, 87.5% sensitivity, and 0.805 AUC for PVT prediction (P < 0.001) at cutoff values of >7.5. Conclusions: This study suggests that increased central obesity and LAR were independently associated with PVT development in non-cirrhotic NAFLD patients, and they should be considered risk factors that may participate in PVT multifactorial pathogenesis.

Liposuction: Drains, Are They Adequate?

Seroma following liposuction (especially mega-sessions; more than 5 L) is a common complication that causes much distress to the surgeon and the patient. This will eventually affect the overall satisfaction and patient's experience regarding liposuction. If not detected promptly, seromas can impair the results. METHODS: This is a prospective analysis performed by the authors in a private practice. All our patients had mega-liposuction sessions (more than 5 L, range 8-12 L) using Power-assisted Liposuction with Lipomatic by Euromai and VASER. Tumescent infiltration was used. Fifty male patients participated after providing their informed consent. Their mean age was 35 years (range, 21-50) and mean body mass index was 29 (range, 28-33). Patients were divided into 2 groups. Group A consisted of 25 patients with no adjunctive draining procedures done, and group B consisted of 25 patients with drainage procedures done. Patients were followed up every other day for 3 weeks for detection of seroma. RESULTS: Seventeen patients had post-operative seroma: 13 in group A and 4 in group B. The volume of seromas was further subdivided into mild (<50 cc), moderate (50-100 cc), and severe (>100 cc). CONCLUSIONS: High definition liposuction is a demanding procedure by both the surgeon and the patient to achieve the best results and contour. Drainage procedures and drains placement are truly effective methods for minimizing seroma formation, enhancing the recovery, and eventually improving the results.

Annexin A6-A multifunctional scaffold in cell motility.

Annexin A6 (AnxA6) belongs to a highly conserved protein family characterized by their calcium (Ca2+)-dependent binding to phospholipids. Over the years, immunohistochemistry, subcellular fractionations, and live cell microscopy established that AnxA6 is predominantly found at the plasma membrane and endosomal compartments. In these locations, AnxA6 acts as a multifunctional scaffold protein, recruiting signaling proteins, modulating cholesterol and membrane transport and influencing actin dynamics. These activities enable AnxA6 to contribute to the formation of multifactorial protein complexes and membrane domains relevant in signal transduction, cholesterol homeostasis and endo-/exocytic membrane transport. Hence, AnxA6 has been implicated in many biological processes, including cell proliferation, survival, differentiation, inflammation, but also membrane repair and viral infection. More recently, we and others identified roles for AnxA6 in cancer cell migration and invasion. This review will discuss how the multiple scaffold functions may enable AnxA6 to modulate migratory cell behavior in health and disease.

Hydroxyquinoline derived vanadium(IV and V) and copper(II) complexes as potential anti-tuberculosis and anti-tumor agents.

Several mixed ligand vanadium and copper complexes were synthesized containing 8-hydroxyquinoline (8HQ) and a ligand such as picolinato (pic(-)), dipicolinato (dipic(2-)) or a Schiff base. The complexes were characterized by spectroscopic techniques and by single-crystal X-ray diffraction in the case of [V(V)O(L-pheolnaph-im)(5-Cl-8HQ)] and [V(V)O(OMe)(8HQ)2], which evidenced the distorted octahedral geometry of the complexes. The electronic absorption data showed the presence of strong ligand to metal charge transfer bands, significant solvent effects, and methoxido species in methanol, which was further confirmed by (51)V-NMR spectroscopy. The structures of [Cu(II)(dipic)(8HQ)]Na and [V(IV)O(pic)(8HQ)] were confirmed by EPR spectroscopy, showing only one species in solution. The biological activity of the compounds was assessed through the minimal inhibitory concentration (MIC) of the compounds against Mycobacterium tuberculosis (Mtb) and the cytotoxic activity against the cisplatin sensitive/resistant ovarian cells A2780/A2780cisR and the non-tumorigenic HEK cells (IC50 values). Almost all tested vanadium complexes were very active against Mtb and the MICs were comparable to, or better than, the MICs of drugs, such as streptomycin. The activity of the complexes against the A2780 cell line was dependent on incubation time presenting IC50 values in the 3-14 µM (at 48 h) range. In these conditions, the complexes were significantly (*P<0.05-**P<0.001) more active than cisplatin (22 µM), in the A2780 cells and even surpassing its activity in the cisplatin-resistant cells A2780cisR (2.4-8 µM vs. 75.4; **P<0.001). In the non-tumorigenic HEK cells poor selectivity toward cancer cells for most of the complexes was observed, as well as for cisplatin.

Safety models incorporating graph theory based transit indicators.

There is a considerable need for tools to enable the evaluation of the safety of transit networks at the planning stage. One interesting approach for the planning of public transportation systems is the study of networks. Network techniques involve the analysis of systems by viewing them as a graph composed of a set of vertices (nodes) and edges (links). Once the transport system is visualized as a graph, various network properties can be evaluated based on the relationships between the network elements. Several indicators can be calculated including connectivity, coverage, directness and complexity, among others. The main objective of this study is to investigate the relationship between network-based transit indicators and safety. The study develops macro-level collision prediction models that explicitly incorporate transit physical and operational elements and transit network indicators as explanatory variables. Several macro-level (zonal) collision prediction models were developed using a generalized linear regression technique, assuming a negative binomial error structure. The models were grouped into four main themes: transit infrastructure, transit network topology, transit route design, and transit performance and operations. The safety models showed that collisions were significantly associated with transit network properties such as: connectivity, coverage, overlapping degree and the Local Index of Transit Availability. As well, the models showed a significant relationship between collisions and some transit physical and operational attributes such as the number of routes, frequency of routes, bus density, length of bus and 3+ priority lanes.