Kaempferol-Enhanced Migration and Differentiation of C2C12 Myoblasts via ITG1B/FAK/Paxillin and IGF1R/AKT/mTOR Signaling Pathways.

SCOPE: Kaempferol (KMP), a bioactive flavonoid compound found in fruits and vegetables, contributes to human health in many ways but little is known about its relationship with muscle mass. The effect of KMP on C2C12 myoblast differentiation and the mechanisms that might underlie that effect are studied. METHODS AND RESULTS: This study finds that KMP (1, 10 µM) increases the migration and differentiation of C2C12 myoblasts in vitro. Studying the possible mechanism underlying its effect on migration, the study finds that KMP activates Integrin Subunit Beta 1 (ITGB1) in C2C12 myoblasts, increasing p-FAK (Tyr398) and its downstream cell division cycle 42 (CDC42), a protein previously associated with cell migration. Regarding differentiation, KMP upregulates the expression of myosin heavy chain (MHC) and activates IGF1/AKT/mTOR/P70S6K. Interestingly, pretreatment with an AKT inhibitor (LY294002) and siRNA knockdown of IGF1R leads to a decrease in cell differentiation, suggesting that IGF1/AKT activation is required for KMP to induce C2C12 myoblast differentiation. CONCLUSION: Together, the findings suggest that KMP enhances the migration and differentiation of C2C12 myoblasts through the ITG1B/FAK/paxillin and IGF1R/AKT/mTOR pathways. Thus, KMP supplementation might potentially be used to prevent or delay age-related loss of muscle mass and help maintain muscle health.

Influence of elevated liver enzyme level on 30-day mortality rates in patients undergoing nonemergency orthopedic surgery.

BACKGROUND: The effect of elevated preoperative liver enzyme levels on postoperative outcomes is a topic of concern to clinicians. This study explored the association between elevated preoperative liver enzyme levels and surgical outcomes in patients undergoing orthopedic surgery. METHODS: Using the American College of Surgeons National Surgical Quality Improvement Program database, we obtained data on adult patients who received nonemergency orthopedic surgery under general anesthesia between 2011 and 2021. RESULTS: We evaluated the data of 477,524 patients, of whom 6.1% (24 197 patients) had elevated preoperative serum glutamic oxaloacetic transaminase (SGOT) levels. An elevated SGOT level was significantly associated with 30-day postoperative mortality (adjusted hazard ratio, 1.62; 95% confidence interval, 1.39 to 1.90). We determined that the mortality rate rose with SGOT levels. The results remained unchanged after propensity score matching. CONCLUSION: Elevated preoperative SGOT levels constitute an independent risk factor for 30-day postoperative mortality and are proportionately associated with the risk of 30-day postoperative mortality.

Potential Therapeutic Improvements in Prostate Cancer Treatment Using Pencil Beam Scanning Proton Therapy with LETd Optimization and Disease-Specific RBE Models.

PURPOSE: To demonstrate the feasibility of improving prostate cancer patient outcomes with PBS proton LETd optimization. METHODS: SFO, IPT-SIB, and LET-optimized plans were created for 12 patients, and generalized-tissue and disease-specific LET-dependent RBE models were applied. The mean LETd in several structures was determined and used to calculate mean RBEs. LETd- and dose-volume histograms (LVHs/DVHs) are shown. TODRs were defined based on clinical dose goals and compared between plans. The impact of robust perturbations on LETd, TODRs, and DVH spread was evaluated. RESULTS: LETd optimization achieved statistically significant increased target volume LETd of ~4 keV/µm compared to SFO and IPT-SIB LETd of ~2 keV/µm while mitigating OAR LETd increases. A disease-specific RBE model predicted target volume RBEs > 1.5 for LET-optimized plans, up to 18% higher than for SFO plans. LET-optimized target LVHs/DVHs showed a large increase not present in OARs. All RBE models showed a statistically significant increase in TODRs from SFO to IPT-SIB to LET-optimized plans. RBE = 1.1 does not accurately represent TODRs when using LETd optimization. Robust evaluations demonstrated a trade-off between increased mean target LETd and decreased DVH spread. CONCLUSION: The demonstration of improved TODRs provided via LETd optimization shows potential for improved patient outcomes.

Development of a mouse model expressing a bifunctional glutathione-synthesizing enzyme to study glutathione limitation in vivo.

Glutathione (GSH) is a highly abundant tripeptide thiol that performs diverse protective and biosynthetic functions in cells. While changes in GSH availability are associated with inborn errors of metabolism, cancer, and neurodegenerative disorders, studying the limiting role of GSH in physiology and disease has been challenging due to its tight regulation. To address this, we generated cell and mouse models that express a bifunctional glutathione-synthesizing enzyme from Streptococcus thermophilus (GshF), which possesses both glutamate-cysteine ligase and glutathione synthase activities. GshF expression allows efficient production of GSH in the cytosol and mitochondria and prevents cell death in response to GSH depletion, but not ferroptosis induction, indicating that GSH is not a limiting factor under lipid peroxidation. CRISPR screens using engineered enzymes further revealed genes required for cell proliferation under cellular and mitochondrial GSH depletion. Among these, we identified the glutamate-cysteine ligase modifier subunit, GCLM, as a requirement for cellular sensitivity to buthionine sulfoximine, a glutathione synthesis inhibitor. Finally, GshF expression in mice is embryonically lethal but sustains postnatal viability when restricted to adulthood. Overall, our work identifies a conditional mouse model to investigate the limiting role of GSH in physiology and disease.

A mouse model to study glutathione limitation in vivo.

Glutathione (GSH) is a highly abundant tripeptide thiol that performs diverse protective and biosynthetic functions in cells. While changes in GSH availability are linked to many diseases, including cancer and neurodegenerative disorders, determining the function of GSH in physiology and disease has been challenging due to its tight regulation. To address this, we generated cell and mouse models that express a bifunctional glutathione-synthesizing enzyme from Streptococcus Thermophilus (GshF). GshF expression allows efficient production of GSH in the cytosol and mitochondria and prevents cell death in response to GSH depletion, but not ferroptosis, indicating that GSH is not a limiting factor under lipid peroxidation. CRISPR screens using engineered enzymes revealed metabolic liabilities under compartmentalized GSH depletion. Finally, GshF expression in mice is embryonically lethal but sustains postnatal viability when restricted to adulthood. Overall, our work identifies a conditional mouse model to investigate the role of GSH availability in physiology and disease.

Robust Design of Effective Allosteric Activators for Rsp5 E3 Ligase Using the Machine Learning Tool ProteinMPNN.

We used the deep learning tool ProteinMPNN to redesign ubiquitin (Ub) as a specific and functionally stimulating/enhancing binder of the Rsp5 E3 ligase. We generated 20 extensively mutated─up to 37 of 76 residues─recombinant Ub variants (UbVs), named R1 to R20, displaying well-folded structures and high thermal stabilities. These UbVs can also form stable complexes with Rsp5, as predicted using AlphaFold2. Three of the UbVs bound to Rsp5 with low micromolar affinity, with R4 and R12 effectively enhancing the Rsp5 activity six folds. AlphaFold2 predicts that R4 and R12 bind to Rsp5's exosite in an identical manner to the Rsp5-Ub template, thereby allosterically activating Rsp5-Ub thioester formation. Thus, we present a virtual solution for rapidly and cost-effectively designing UbVs as functional modulators of Ub-related enzymes.

Intra-Articular Lactate Dehydrogenase A Inhibitor Oxamate Reduces Experimental Osteoarthritis and Nociception in Rats via Possible Alteration of Glycolysis-Related Protein Expression in Cartilage Tissue.

Osteoarthritis (OA) is the most common form of arthritis and joint disorder worldwide. Metabolic reprogramming of osteoarthritic chondrocytes from oxidative phosphorylation to glycolysis results in the accumulation of lactate from glycolytic metabolite pyruvate by lactate dehydrogenase A (LDHA), leading to cartilage degeneration. In the present study, we investigated the protective effects of the intra-articular administration of oxamate (LDHA inhibitor) against OA development and glycolysis-related protein expression in experimental OA rats. The animals were randomly allocated into four groups: Sham, anterior cruciate ligament transection (ACLT), ACLT + oxamate (0.25 and 2.5 mg/kg). Oxamate-treated groups received an intra-articular injection of oxamate once a week for 5 weeks. Intra-articular oxamate significantly reduced the weight-bearing defects and knee width in ACLT rats. Histopathological analyses showed that oxamate caused significantly less cartilage degeneration in the ACLT rats. Oxamate exerts hypertrophic effects in articular cartilage chondrocytes by inhibiting glucose transporter 1, glucose transporter 3, hexokinase II, pyruvate kinase M2, pyruvate dehydrogenase kinases 1 and 2, pyruvate dehydrogenase kinase 2, and LHDA. Further analysis revealed that oxamate significantly reduced chondrocyte apoptosis in articular cartilage. Oxamate attenuates nociception, inflammation, cartilage degradation, and chondrocyte apoptosis and possibly attenuates glycolysis-related protein expression in ACLT-induced OA rats. The present findings will facilitate future research on LDHA inhibitors in prevention strategies for OA progression.

Anatomical changes in the male pelvis between the supine and upright positions-A feasibility study for prostate treatments in the upright position.

Treating and imaging patients in the upright orientation is gaining acceptance in radiation oncology and radiology and has distinct advantages over the recumbent position. An IRB approved study to investigate the positions and orientations of the male pelvic organs between the supine and upright positions was conducted. The study comprised of scanning 15 male volunteers (aged 55-75 years) on a 0.6 T Fonar MRI scanner in the supine and upright positions with a full bladder and in the upright position with an empty bladder. The Pelvic study revealed that in the upright position the 1. Position and shape of the prostate are not impacted significantly by bladder fill. 2. Distance between the sacrum and the anterior bladder wall is significantly smaller. 3. Anterior-Posterior length and the bladder width is significantly larger. 4. Seminal vesicles are pushed down by the bladder. 5. Top of the penile bulb is further away from the apex of the prostate. These observed differences could positively impact upright prostate treatments by 1. Reducing the risk of small bowel approximating the treatment volume. 2. Prostate treatments can be done with a reduced focus on bladder fill. 3. Radiation beams for treating intermediate risk prostrate can be made smaller or a larger portion of the seminal vesicles can be treated with the same beam size than typically used for supine treatments. 4. Reducing the average dose to the penile bulb.

Isoaaptamine increases ROS levels causing autophagy and mitochondria-mediated apoptosis in glioblastoma multiforme cells.

Glioblastoma multiforme (GBM) is a common central nervous system disease with a poor prognosis; its five-year survival rate is <5 %, and its median survival of 15 months. Current treatment includes chemotherapy with temozolomide, which is ineffective against GBM, suggesting an urgent need to develop novel therapies. This study evaluated isoaaptamine and aaptamine in the GBM cell lines for cell viability; GBM 8401, U87 MG, U138 MG, and T98G. Our findings showed that isoaaptamine was more potent than its iso-form aaptamine in these four cell lines, and GBM 8401 was most sensitive to isoaaptamine. The study in GBM 8401 cells showed that apoptosis was induced by isoaaptamine with increased cleaved caspase 3 and poly ADP-ribose polymerase (PARP). Moreover, isoaaptamine enhanced oxidative stress by increasing the levels of reactive oxygen species (ROS), inhibiting mitochondrial and cellular superoxidase dismutases (SOD1&2), peroxidase and an anti-apoptotic protein (Bcl-2), and disrupting mitochondrial membrane potential. In addition, the oxygen consumption rates and activities of mitochondrial complexes I-V were significantly reduced. Mitochondrial dynamics were prone to fission instead of fusion after isoaaptamine treatment, and ATP synthesis was ablated. Also, autophagy-related acidic organelle vesicles were formed, indicating autophagy was triggered. Overall, isoaaptamine-induced ROS overproduction in mitochondria could cause mitochondrial dysfunction, apoptosis, and autophagy in the GBM cells.

Effect of dietary supplementation of Bacillus subtilis TLRI 211-1 on laying performance, egg quality and blood characteristics of Leghorn layers.

OBJECTIVE: TLRI 211-1 is a novel Bacillus subtilis strain. This experiment was to investigate dietary supplementation of TLRI 211-1 on laying performance, egg quality and blood characteristics of layers. METHODS: One hundred and twenty 65-wk-old Leghorn layers were divided into four treatment groups for 8 weeks experiment. Each treatment had three replicates. The basal diet was formulated as control group with crude protein 17% and metabolizable energy 2,850 kcal/kg and supplemented with TLRI 211-1 0.1%, 0.3%, and commercial Bacillus amyloliquefaciens 0.1% as treatment 2, 3 and 4 groups, respectively. Both TLRI 211-1 and commercial Bacillus amyloliquefaciens were adjusted to contain 1×109 colony-forming unit (CFU)/mL (g), hence the 0.1% supplemental level was 1×109 CFU/kg. RESULTS: The results showed that TLRI 211-1 0.3% and commercial B. amyloliquefaciens groups had higher weight gain than the other groups; TLRI 211-1 0.1% group had better feed to eggs conversion ratio than the control and commercial B. amyloliquefaciens groups (p<0.05). Bacillus subtilis supplementation increased yolk weight (p<0.05). In egg quality during storage, TLRI 211-1 0.1% had higher breaking strength than the control group at the second week of storage (p<0.05). At the third week of storage, TLRI 211-1 0.3% had higher Haugh unit (p<0.05). Hens fed diets supplemented with TLRI 211-1 0.3% significantly decreased blood triglyceride levels and increased blood calcium levels (p< 0.05). TLRI 211-1 0.3% group had lower H2S (p<0.05) and hence had less unpleasant odor in excreta of hens. CONCLUSION: In conclusion, supplementation with 0.1% TLRI 211-1 can significantly improve feed to eggs conversion ratio. TLRI 211-1 supplementation also can maintain eggs at their optimum quality level during storage. The study showed that B. subtilis TLRI 211-1 can be used as feed additives for improving egg production performance and egg quality.

Gamma Frequency Inhibits the Secretion and Aggregation of Amyloid-ß and Decreases the Phosphorylation of mTOR and Tau Proteins in vitro.

BACKGROUND: Alzheimer's disease (AD) was the main cause of dementia in an aging society; unfortunately, there is no effective treatment for AD now. Meditation has been reported to thicken the cerebral cortex, and gamma wave at a frequency of 40 hertz (Hz) was recorded during the meditation process from the brain. Previous study showed that non-invasive scintillation gamma frequency oscillation increased the space in recognition and memory of auditory cortex hippocampal gyrus in AD mice model. However, the AD-related molecular change by exposure of 40 Hz gamma frequency in brain cells was still unclear. OBJECTIVE: We investigated the AD-related molecular change by exposure of 40 Hz gamma frequency in SH-SY5Y cells. METHODS: We designed the light and sound generators at 40 Hz gamma frequency for this study. SH-SY5Y cells were exposed to sound or light of 40 Hz gamma frequency, respectively. The concentrations of amyloid-ß40 (Aß40) and amyloid-ß42 (Aß42) were quantified by enzyme-linked immunosorbent assay. The protein levels were examined by Western blotting. The aggregation of Aß42 was examined by thioflavin T assay. RESULTS: Our results showed that the secretion of Aß, phosphorylation of AKT, mTOR, and tau, and aggregation of Aß42 were significantly inhibited by 40 Hz gamma frequency in SH-SY5Y cells. The phosphorylation of 4E-BP1, downstream of mTOR, was induced by 40 Hz gamma frequency in SH-SY5Y cells. CONCLUSION: Our study showed 40 Hz gamma frequency involved in the inhibition of secretion and aggregation of Aß and inhibition of p-Tau protein expression through the mTOR/4E-BP1/Tau signaling pathway.

The Promotion of Migration and Myogenic Differentiation in Skeletal Muscle Cells by Quercetin and Underlying Mechanisms.

Aging and muscle disorders frequently cause a decrease in myoblast migration and differentiation, leading to losses in skeletal muscle function and regeneration. Several studies have reported that natural flavonoids can stimulate muscle development. Quercetin, one such flavonoid found in many vegetables and fruits, has been used to promote muscle development. In this study, we investigated the effect of quercetin on migration and differentiation, two processes critical to muscle regeneration. We found that quercetin induced the migration and differentiation of mouse C2C12 cells. These results indicated quercetin could induce myogenic differentiation at the early stage through activated p-IGF-1R. The molecular mechanisms of quercetin include the promotion of myogenic differentiation via activated transcription factors STAT3 and the AKT signaling pathway. In addition, we demonstrated that AKT activation is required for quercetin induction of myogenic differentiation to occur. In addition, quercetin was found to promote myoblast migration by regulating the ITGB1 signaling pathway and activating phosphorylation of FAK and paxillin. In conclusion, quercetin can potentially be used to induce migration and differentiation and thus improve muscle regeneration.

2.2 Å Cryo-EM Tetra-Protofilament Structure of the Hamster Prion 108-144 Fibril Reveals an Ordered Water Channel in the Center.

Fibrils of the hamster prion peptide (sHaPrP, sequence 108-144) were prepared in an acidic solution, and their structure was solved by cryogenic electron microscopy with a resolution of 2.23 Å based on the gold-standard Fourier shell correlation (FSC) curve. The fibril has a novel architecture that has never been found in other amyloid fibrils. Each fibril is assembled by four protofilaments (PFs) and has an ordered water channel in the center. Each protofilament contains three ß-strands (125-130, 133-135, and 138-141) arranged in an "R"-shaped construct. The structural data indicate that these three ß-strand segments are the most amyloidogenic region of the prion peptide/protein and might be the site of nucleation during fibrillization under conditions without denaturants.

Chlorogenic Acid Inhibition of Esophageal Squamous Cell Carcinoma Metastasis via EGFR/p-Akt/Snail Signaling Pathways.

BACKGROUND/AIM: Chlorogenic acid (CGA) is a polyphenol compound found in a variety of foods, including coffee, tea, cherries, and apples. It has been found by a number of studies to affect the viability of human cancer cells. No study has investigated its effect on esophageal squamous cell carcinoma (ESCC) metastasis or the molecular mechanism underlying its effect on this disease. MATERIALS AND METHODS: We first used the Taiwanese ESCC cell line CE81T/VGH to create CE81T-M4 cells. Treatment of higher motility cells with chlorogenic acid for 24 h led to inhibition of cell migration and invasion as shown by scratch migration and transwell assays. RESULTS: Western blotting showed that chlorogenic acid halted the activation of EGFR/p-Akt/Snail pathway and suppressed the expression of MMP-2 and MMP-9. Knockdown of either EGFR or Akt inhibited Snail, MMP2, and MMP9 activity as well as cell migration and invasion. CONCLUSION: Chlorogenic acid inhibited cancer cell motility via the EGFR/p-Akt/Snail pathway and could potentially be used to develop an antimetastatic agent for ESCC in the future.

Metabolic-scale gene activation screens identify SLCO2B1 as a heme transporter that enhances cellular iron availability.

Iron is the most abundant transition metal essential for numerous cellular processes. Although most mammalian cells acquire iron through transferrin receptors, molecular players of iron utilization under iron restriction are incompletely understood. To address this, we performed metabolism-focused CRISPRa gain-of-function screens, which revealed metabolic limitations under stress conditions. Iron restriction screens identified not only expected members of iron utilization pathways but also SLCO2B1, a poorly characterized membrane carrier. SLCO2B1 expression is sufficient to increase intracellular iron, bypass the essentiality of the transferrin receptor, and enable proliferation under iron restriction. Mechanistically, SLCO2B1 mediates heme analog import in cellular assays. Heme uptake by SLCO2B1 provides sufficient iron for proliferation through heme oxygenases. Notably, SLCO2B1 is predominantly expressed in microglia in the brain, and primary Slco2b1-/- mouse microglia exhibit strong defects in heme analog import. Altogether, our work identifies SLCO2B1 as a microglia-enriched plasma membrane heme importer and provides a genetic platform to identify metabolic limitations under stress conditions.

The effect of a multidisciplinary lifestyle modification program for obese and overweight children.

BACKGROUND/PURPOSE: The increasing prevalence of overweight and obese children and adolescents has been recognized as a public health threat worldwide. This study aimed to assess the effect of a stepwise lifestyle intervention in children and adolescents. METHODS: We developed a multidisciplinary clinic aimed at providing lifestyle interventions for obese children and adolescents. The program comprised three stages with stepwise goals: knowledge building (the first 4 weeks), habit consolidation (5-12 weeks), and self-monitoring (13-20 weeks). RESULTS: Of the 63 participants (age 11.6 ± 3.2 years) who entered the first stage of the program, 48, 22, and 15 completed the first, second and third stages (4, 12, and 20 weeks), respectively. In the first stage, significant improvement was noted in body weight, body mass index (BMI), BMI z-score, and waist circumference. Improvements in physical fitness performance were observed at 4 weeks in 3/5 items and at 12 weeks in 4/5 items. The decreases in body weight, BMI and BMI z-score were most prominent in the first two stages. In the third stage, participants maintained a stable body weight. In the 15 subjects who completed the whole program, BMI decreased from 29.3 ± 6.9 to 27.8 ± 6.1 (P = 0.001), and BMI z-score decreased from 3.06 ± 0.96 to 2.69 ± 0.91(P = 0.001). CONCLUSION: We developed a feasible multidisciplinary program based on knowledge education and individualized training. BMI and physical fitness scores can be used as early indicators of lifestyle change for obese children and adolescents.

SLC25A39 is necessary for mitochondrial glutathione import in mammalian cells.

Glutathione (GSH) is a small-molecule thiol that is abundant in all eukaryotes and has key roles in oxidative metabolism1. Mitochondria, as the major site of oxidative reactions, must maintain sufficient levels of GSH to perform protective and biosynthetic functions2. GSH is synthesized exclusively in the cytosol, yet the molecular machinery involved in mitochondrial GSH import remains unknown. Here, using organellar proteomics and metabolomics approaches, we identify SLC25A39, a mitochondrial membrane carrier of unknown function, as a regulator of GSH transport into mitochondria. Loss of SLC25A39 reduces mitochondrial GSH import and abundance without affecting cellular GSH levels. Cells lacking both SLC25A39 and its paralogue SLC25A40 exhibit defects in the activity and stability of proteins containing iron-sulfur clusters. We find that mitochondrial GSH import is necessary for cell proliferation in vitro and red blood cell development in mice. Heterologous expression of an engineered bifunctional bacterial GSH biosynthetic enzyme (GshF) in mitochondria enables mitochondrial GSH production and ameliorates the metabolic and proliferative defects caused by its depletion. Finally, GSH availability negatively regulates SLC25A39 protein abundance, coupling redox homeostasis to mitochondrial GSH import in mammalian cells. Our work identifies SLC25A39 as an essential and regulated component of the mitochondrial GSH-import machinery.

Fast MCsquare-Based Independent Dose Verification Platform for Pencil Beam Scanning Proton Therapy.

PURPOSE: To commission MCsquare (a multi-cores CPU-based dose calculation engine) for pencil beam scanning (PBS) proton therapy, integrate it into RayStation treatment plan system (TPS) to create a dedicated platform for fast independent dose verification. METHOD: A MCsquare-based independent dose verification platform (MC2InRS) was developed to realize automatic dose re-calculation for clinical use, including data preparation, dose calculation, 2D/3D gamma analysis. MCsquare was commissioned based on in-air lateral dose profiles, integrated depth dose, and the absolute dose of different beam energies for Proteus®ONE. MC2InRS was validated with measurement data using various targets and depths in a water phantom. This study also investigated 15 clinical cases to demonstrate the feasibility and effectiveness of MC2InRS platform in clinic practice. RESULTS: Between simulation and measurement, the distal range differences at 80% (R80) and 20% (R20) dose levels for each energy were below 0.05 mm, and 0.1 mm, respectively, and the absolute dose differences were below 0.5%. 29 out of 36 QA planes reached a 100% gamma passing rate (GPR) for 2%/2mm criteria, and a minimum of 98.3% gamma was obtained in water phantom between simulation and measurement. For the 15 clinical cases investigated, the average 2D GPR (2%/2mm) was 95.4%, 99.3% for MCsquare vs. measurement, MCsquare vs. TPS, respectively. The average 3D GPR (2%/2mm) was 98.9%, 95.3% for MCsquare vs. TPS in water, and computed tomography (CT), respectively. CONCLUSION: MC2InRS, a fast, independent dose verification platform, has been developed to perform dose verification with high accuracy and efficiency for Pencil Bream Scanning (PBS). Its potential to be applied in routine clinical practice has also been discussed.

The root cause analysis on failed patient-specific measurements of pencil beam scanning protons using a 2D detection array with finite size ionization chambers.

The aim of this report is to present the root cause analysis on failed patient-specific quality assurance (QA) measurements of pencil beam scanning (PBS) protons; referred to as PBS-QA measurement. A criterion to fail a PBS-QA measurement is having a <95% passing rate in a 3.0%-3.0 mm gamma index analysis. Clinically, we use a two-dimensional (2D) gamma index analysis to obtain the passing rate. The IBA MatriXX PT 2D detection array with finite size ionization chamber was utilized. A total of 2488 measurements performed in our PBS beamline were cataloged. The percentage of measurements for the sites of head/neck, breast, prostate, and other are 53.3%, 22.7%, 10.5%, and 13.5%, respectively. The measurements with a passing rate of 100 to >94%, 94 to >88%, and <88% were 93.6%, 5.6%, and 0.8%, respectively. The percentage of failed measurements with a <95% passing rate was 10.9%. After removed the user errors of either re-measurement or re-analysis, 8.1% became acceptable. We observed a feature of >3% per mm dose gradient with respect to depth on the failed measurements. We utilized a 2D/three-dimensional (3D) gamma index analysis toolkit to investigate the effect of depth dose gradient. By utilizing this 3D toolkit, 43.1% of the failed measurements were improved. A feature among measurements that remained sub-optimal after re-analysis was a sharp >3% per mm lateral dose gradient that may not be well handled using the detector size of 5.0 mm in-diameter. An analysis of the sampling of finite size detectors using one-dimensional (1D) error function showed a large dose deviation at locations of low-dose areas between two high-dose plateaus. User error, large depth dose gradient, and the effect of detector size are identified as root causes. With the mitigation of the root causes, the goals of patient-specific QA, specifically detecting actual deviation of beam delivery or identifying limitations of the dose calculation algorithm of the treatment planning system, can be directly related to failure of the PBS-QA measurements.