Gout flares have been challenging to identify in retrospective databases due to gout flares not being well documented by diagnosis codes, making it difficult to conduct accurate database studies. Previous studies have used different algorithms, and in this study, we used a computer-based method to identify gout flares. The objectives of this study were to identify gout flares in gout patients newly initiated on urate-lowering therapy and evaluate factors associated with a patient experiencing gout flares after starting drug treatment. This was a retrospective cohort study identifying gout patients newly initiated on a urate-lowering therapy (ULT) during the study time period of January 1, 2007–December 31, 2010. The index date was the first dispensed ULT prescription during the study time period. Patients had to be ≥18 years of age on index date, have no history of prior ULT prescription during 12 months before index date, and were required to have 12 months of continuous membership with drug benefit during pre-/post-index. Electronic chart notes were reviewed to identify gout flares; these reviews helped create a validated computer-based method to further identify patients with gout flares and were categorized into 0 gout flares, 1–2 gout flares, and ≥3 gout flares during the 12 months post-index period. Multivariable logistic regression was used to examine patient and clinical factors associated with gout flares during the 12-month follow-up period. There were 8905 patients identified as the final cohort and 68 % of these patients had one or more gout flares during the 12-month follow-up: 2797 patients (31 %) had 0 gout flares, 4836 (54 %) had 1–2 gout flares, and 1272 patients (14 %) had ≥3 gout flares. Using a multivariate regression analyses, factors independently associated with 1–2 gout flares and ≥3 gout flares versus no gout flares were similar, however, with slight differences, such as younger patients were more likely to have 1–2 gout flares and patients ≥65 years of age had ≥3 gout flares. Factors such as male gender, not attaining sUA goal, having ≥3 comorbidities, diuretics use, no changes in initial ULT dose, and not adhering to ULT all were associated with gout flares versus no gout flares. Using a new method to identify gout flares, we had the opportunity to compare our findings with the previous studies. Our study findings echo other previous studies where older patients, male, diuretics, having a greater number of comorbidities, and non-adherence are more likely to have more gout flares during the first year of newly initiating ULT. There is an unmet need for patients with gout to be educated and managed more closely, especially during the first year. Acute gout flare is the most common manifestation of gout and has been described as an acute inflammatory reaction with red, swollen, and painful joints , 2. GoutGout flares can be challenging to identify in clinical databases due to gout flares not being well documented by diagnosis codes, thus making it difficult to conduct accurate retrospective studies. Previous studies have used different algorithms consisting of diagnosis codes, radiology reports, pharmacy, and medical claims to identify gout flares , 17, 18.Kaiser Permanente Southern California (KPSC) provides integrated, comprehensive medical services to 3.6 million members through its own facilities, which includes 14 hospitals, 202 outpatient facilities, and a centralized laboratory. Every member receives a unique medical record number that they keep for life. This allows the member to be linked to various clinical and administrative databases including member enrollment and benefits, inpatient and outpatient visits, laboratory test results, and drug dispensing. All aspects of care and interactions with the healthcare delivery system are identified in a comprehensive electronic medical record (EMR) system. In addition, care delivered outside KPSC is identified by a claims system. The KPSC membership represents 15 % of the population in the Southern California region and closely mirrors the Southern California population; it is racially diverse and includes the entire socioeconomic spectrum , 22. The2, history of dialysis, active cancer, current chemotherapy, or kidney stones/nephrolithiasis  during the study time period; the index date was defined as the patient’s first ULT prescription identified during the study time period. Patients had to be ≥18 years of age at time of index date and were required to have at least 12 months of KPSC membership eligibility including drug benefits prior to their index date, index date, and 12 months post-index. Enrollment gaps of ≤30 days were considered continuous enrollment. Eligible patients were required to have two outpatient gout diagnoses  ≥30 days apart or one inpatient gout diagnosis code in any position anytime during the study time period. We identified patients newly initiating an ULT prescription if they had no ULT prescription during the 12 months prior to their index date. Patients were excluded if they had history of human immunodeficiency virus (HIV), a diagnosis code for chronic kidney disease (CKD) stage 5 or an estimated glomerular filtration rate (GFR) <15 ml/min/1.73 mIn previous studies , 17, 18,Baseline characteristics (during the 12 months prior to the index date and index date) included age, sex, race, comorbid conditions, anti-inflammatory plus other concomitant medication use, initial dose of ULT, sUA levels, renal function, and prescriber specialty. Comorbid conditions included alcohol use, cardiovascular disease, diabetes, hypertension, other arthritic conditions, and obesity. Baseline GFR levels for renal function and baseline sUA levels were measured up to 12 months prior to the index date, and if patients had multiple baseline sUA levels available, the measurement obtained most proximate to the index date was used. Prescription anti-inflammatory medications  and concomitant medications  were all identified from electronic dispensing records. The initial dose of ULT was calculated using pharmacy data which included quantity number, strength, day’s supply, and directions from the dispensed prescription. Prescriber specialty was determined at the time of the index dispensed ULT prescription and was categorized as primary care, rheumatologist, or other.Outcomes of interest were identified during post-index for each of the gout flare groups. Outcomes included changes in ULT therapy from baseline such as dose escalation, dose equal, and dose decrease; adherence to ULT being measured using proportion of days covered (PDC) method; utilization of anti-inflammatory therapies during post-index; and sUA goal attainment were identified during post-index. The sUA levels were evaluated for patients that had a baseline and follow-up level. Patients were considered to have had ULT dose escalation if the final observed daily dose was greater than the index dose. Conversely, patients were considered to have had a dose decrease if the final daily ULT dose was less than the initial dose. If there was no change in the ULT dose, then it remained dose equal. PDC was calculated as the number of days with ULT drug dispensed divided by the number of days in the specified time interval (365 days). We evaluated the PDC within the first 12 months of initiating ULT. The PDC was dichotomized <80 % considered as non-adherent and ≥80 % considered adherent. Attainment of sUA goal was defined as the last follow-up sUA obtained after the index date with a value <6.0 mg/dl.t test for continuous variables and the Chi-squared statistic for categorical variables. Multivariable logistic regression models were used to evaluate factors associated with patients having 1–2 gout flares versus patients with 0 gout flares and patients with ≥3 gout flares versus patients with 0 gout flares. Factors including age, sex, race, severity of comorbid conditions, diuretic use, prescriber specialty, and anti-inflammatory medications were controlled for in the models. All data were analyzed using SAS version 9.2 . p values <0.05 were considered to be statistically significant.Unadjusted descriptive statistics summarized patient characteristics of the study population where patients with 0 gout flares were compared to patients with 1–2 gout flares and patients with 0 gout flares were compared to patients with ≥3 gout flares. Differences between these patients groups were tested using two-sided There were 8828 gout patients identified as newly initiated on a ULT, using our selection criteria Fig. ; 68 % ofTable Using multivariate logistic regression analysis, we identified patient and clinical factors that were independently associated with patients who were having gout flares versus patients who were not having gout flares Table . FactorsThis is the first study to identify gout flares utilizing a novel validated NLP + ML computer-based algorithm using text searches of clinical notes in EMR. This method provided results with a much higher sensitivity and specificity, when compared to the other database algorithm methods: 82.1 % sensitivity, 91.5 % specificity, 77.9 % positive predictive value (PPV), and 93.4 % negative predictive value (NPV) for identifying gout flares at the note level . CompareGout is associated with many comorbidities, such as obesity, diabetes, renal insufficiency , 23–28, The relationship between high sUA levels and the recurrent gout flares has been shown in previous studies –5. ConsiAs with any retrospective study, there are some limitations that need to be addressed. One is investigator dependence on the availability and accuracy of the pharmacy and medical records to identify the study population. To help alleviate this concern, we identified patients with gout using two gout diagnoses ≥30 days apart or one inpatient gout diagnosis code, in addition to a prescription for urate-lowering therapy. Another limitation is that the NLP may not be able to identify all patients with acute gouty flares even with the search criteria being established, since gout flares are sometimes self-managed at home or that gout flares are scantly documented. Our validated algorithm with 82.1 % sensitivity and 91.5 % specificity was shown to be superior to traditional methods of identifying gout . AnotherIn this study, we used a new and different method to identify gout flares. The validated computer-based method produced findings consistent with other studies, where patients older in age, male, not attaining sUA goal of <6 mg/dl, non-adherence to ULT, diuretic use, and more comorbidities are associated with more gout flares during the first year of newly initiating ULT. Patient education and physician involvement are two keys to reduction in gout flares, especially during the first year where most gout flares occur from initiation of ULT. Patients who follow the recommendations to initiate ULT with a low starting dose or increase the ULT slowly tend to have fewer adverse events , 33. Cur


Our theoretical study offers a new paradigm for understanding the mechanisms of the ultra-high spatial resolution demonstrated in tip-enhanced spectroscopy which is of importance but neglected.Tip-enhanced Raman spectroscopy (TERS) with sub-nanometer spatial resolution has been recently demonstrated experimentally. However, the physical mechanism underlying is still under discussion. Here we theoretically investigate the electric field gradient of a coupled tip-substrate system. Our calculations suggest that the ultra-high spatial resolution of TERS can be partially attributed to the electric field gradient effect owning to its tighter spatial confinement and sensitivity to the infrared (IR)-active of molecules. Particularly, in the case of TERS of flat-lying H Tip-enhanced Raman spectroscopy (TERS) is a particularly important technique for molecule analysis on the nanometer scale because of its high detection sensitivity and spatial resolution1234568910111317182TBPP molecules and H2TBPP molecules with a small tilted angle, the electric field gradient has a larger effect than the electric field in terms of the spectral spatial resolution. The calculated spectral spatial resolution, Raman and infrared spectra coincide with the experimental results indicating the strong influence of electric field gradient on the tip-enhanced spectroscopyHere we show by theoretical calculations that for flat-lying Hin situ1320αβα, Aα,βγ and αβ,γδC are the electric dipole-dipole polarizability, dipole-quadrupole polarizability and quadrupole-quadrupole polarizability, respectively, describing the distortion of the molecule by an external electric field. αE, βE and γE are the external electric field. α,βγ and αβ,γδC account for the electric field gradient multiple Raman corresponding to IR-active modes2324In tip-enhanced Raman spectroscopy, both Raman-active modes and infrared (IR)-active modes can be simultaneously observed H1 = H2 is omitted. Only the first two terms are big enough to be taken into account even in the high electric field gradient regions. Therefore, the intensity ratio of Raman-active mode to IR-active modes can be written asAccording to To study in detail the electric field enhancement and the electric gradient enhancement in TERS, we perform a numerical simulation, where a model of a conical gold tip on a silver substrate is employed. The exact TERS configuration is shown in loc and the incident field Ein amplitude, |M| = |Eloc/Ein|) at the center of the nanogap between the tip and the substrate as a function of incident wavelength and cone angle of the tip. In all cases we observe two resonance behaviors which are attributed to the vertical (long wavelength) and the horizontal (short wavelength) dipole resonance respectively , and (|Mxy|) respectively, on the wavelength and cone angle of the tip. The plasmon resonances properties for both orientations are similar to that of the total electric field. It should be noted that the maximum of the two quantities are not from the same position. The maximum horizontal electric field enhancement is located within a ring shape, while the maximum vertical electric field enhancement is located at the center of the 2D plane that can be demonstrated in the following section of the paper and the We choose this tip (ϕ = 20°) and horizontal dipole resonance, which coincides with the vibration orientation of flat-lying Hxy|) forms a ring shape with 0.7 < r < 1.7 nm (r is the distance away from the center of the plane) which has a maximum value around 1 × 102. In In 1, I2 and I1/I2 plotted as function of the lateral displacement of the midway on the 2D plane. The spatial resolution is defined as the full width at half maximum (FWHM) in these profiles. As shown in 1is 8.0 × 107 and the spatial resolution can be as high as 1 nm. 2 is 4.7 × 107 and the spatial resolution is 1 nm. In 1 to I2 is 1.8 located at r = ± 1.8 nm. Importantly, the ratio I1/I2 is less than 1 at −0.4 < r < 0.4 nm, which reveals a more contribution of electric field gradient to the ultra-high spatial resolution of TERS than the electric field in the spatial extent of 0.8 nm in diameter directly below the tip, even though the same TERS resolutions of 1 nm are obtained from the two fields. If considering the combined effect of these two plasmon fields, we should obtain an even higher spatial resolution below 1 nm in TERS experiments.It is generally known that the electric field enhancement strongly depends on the tip-substrate distance in TERS system132TBPP molecules. Base on the electric field gradient effect, it is very helpful to understand the underlying physical mechanism of TERS, and obtain the highly resolved optical images of nanometric objects by reasonable optimal design.It has been long believed that the strongest Raman signals of molecules can be achieved when the polarized direction of the external excitation electric field is in line with the vibration direction of the molecules. However, as these results demonstrate, the horizontal electric field gradient could have a more dominant contribution to the TERS resolution than the electric field for flat-lying H2TBPP molecules by Dong group from Ref.1, where the tilt angle of the molecule is 30° and 0°, respectively. Density functional theory (DFT) calculations were performed to obtain the Raman- and IR- active modes of a single H2TBPP molecule in free space, as shown in 2TBPP molecules is stronger than that for molecules perpendicularly adsorbed on the substrate. That agrees well with the variation trend in We provide more evidence of the important contribution of electric field gradient by showing the existence of infrared active modes of molecules in the TERS spectroscopy. 2TBPP molecules. If considering the contribution of vertical electric field and electric field gradient, the spatial resolutions are within 2 nm , which numerically solves the Maxwell's equations2TBPP molecule. The calculations of vibrational spectra of H2TBPP were carried out, using the Gaussian 09 software, the B3LYP functional and 6–31G(d) basis set.To provide more evidence of the important contribution of electric field gradient to the ultra-high spatial resolution of TERS by showing the existence of infrared active modes of molecules, density functional theory (DFT) calculations were performed to obtain the vibrational modes of a single HJ.C., Z.Y. and M.S. supervised the project. M.S., Z.Y. and J.C. proposed the idea. L.M. and M.S. performed the calculations. M.S., L.M., Z.Y. and J.C. analyzed data. M.S. and L.M. wrote the paper. All authors were involved in the discussion and revision of the manuscript.supporting information


This study investigated the epigenetic alteration and biological function of the pro-apoptotic gene ASC/TMS1 in renal cell carcinoma. ASC/TMS1 was downregulated in five out of six RCC cell lines. A significant downregulation was also detected in sixty-seven paired renal tumors compared with adjacent non-cancerous tissues. The downregulation of ASC/TMS1 was correlated with promoter hypermethylation and could be restored with demethylation treatment. Re-expression of ASC/TMS1 in silenced RCC cell lines inhibited cell viability, colony formation, arrested cell cycle, induced apoptosis, suppressed cell invasion and repressed tumorigenicity in SCID mice. The antitumorigenic function of ASC/TMS1 in renal cancer was partially regulated by activation of p53 and p21 signaling. In addition, restoration of ASC/TMS1 sensitizes RCC cells to DNA damaging agents. Knockdown of ASC/TMS1 reduced DNA damaging agents-induced p53 activation and cell apoptosis. Moreover, ASC/TMS1 hypermethylation was further detected in 41.1% (83/202) of RCC tumors, but only 12% in adjacent non-cancerous tissues. ASC/TMS1 methylation was significantly correlated with higher tumor nuclear grade. In conclusion, ASC/TMS1 is a novel functional tumor suppressor in renal carcinogenesis. ASC/TMS1 tumor specific methylation may be a useful biomarker for designing improved diagnostic and therapeutic strategies for RCC. Renal cell carcinoma (RCC) is the most lethal type of urological cancers due to its occult onset and resistance to radiation and chemotherapy, accounting for approximately 3% of all adult malignancies . HoweverGenetic and epigenetic modifications of apoptotic genes was previously reported to affect the development, inflammatory response, carcinogenesis, tumor growth, and chemoradiosensitivity. TMS1 (target of methylation induced silencing), also known as ASC (apoptosis speck-like protein containing a CARD) has been described in the literature as a pro-apoptotic gene which encodes a 22 kDa protein containing a pyrin domain (PYD) in the N-terminus and a caspase recruitment domain (CARD) in the C-terminus. Both PYD and CARD are members of the 6-helix bundle death domain-fold superfamily and are involved in the regulation of apoptosis, nuclear factor-κB activation, and the maturation of proinflammatory cytokines  and ConwBy semiquantitative RT-PCR, we first analyzed the mRNA expression status of ASC/TMS1 in six RCC cell lines, which showed that ASC/TMS1 was silenced or downregulated in five out of six (83.3%) renal cancer cell lines Figure . HoweverTo explore whether promoter methylation directly mediates ASC/TMS1 reduction in RCC, four methylated cell lines  were treated with the DNA methyltransferase inhibitor 5-Aza-2′-deoxycytidine with or without the histone deacetylase inhibitor Trichostatin A. After treatment, ASC/TMS1 expression was significantly increased in these cell lines along with an increase in unmethylated promoter alleles and a decrease in methylated alleles Figure . FurtherP = 0.0001). Inhibition of ASC/TMS1 mRNA expression in the carcinoma tissues of renal cancer patients was further confirmed at protein level by using immunohistochemical staining. We examined ASC/TMS1 protein expression in 67 paired primary RCCs. In adjacent nontumor tissues, intense immunostaining for ASC/TMS1 was observed in a cytoplasmic and nucleus distribution .The mRNA expression of ASC/TMS1 was evaluated in sixty-seven paired primary RCCs using quantitative Real-Time PCR. As shown in Figure n Figure , whereasn Figure . Statistp = 0.005), whereas no significant correlation was found between its methylation and gender, age, tumor location, TNM stage and histological type. These data indicate that ASC/TMS1 methylation is a frequent event in pathogenesis of RCC and is associated with patient poor prognosis.We further analyzed ASC/TMS1 methylation status in paired primary RCC samples and their adjacent nontumor tissues. Of 202 tumor samples 83 (41.1%) showed methylation, but only 12% (3/25) in adjacent non-malignant renal tissues, suggesting tumor-specific methylation of ASC/TMS1 in RCC. Representative methylation status of ASC/TMS1 in RCC primary tumors (T) and paired adjacent nontumor tissues (N) are shown in Figure The frequent silencing of ASC/TMS1 mediated by promoter hypermethylation in RCC, but not in normal renal tissue, suggested that ASC/TMS1 may be a candidate tumor suppressor in renal carcinogenesis. We thus examined the growth inhibitory effect through ectopic expression of ASC /TMS1 in silenced renal cancer cell lines 786–0 and A498. Restored expression of ASC/TMS1 was evidenced by western blot Figure , which dWe investigated the effects of ASC/TMS1 on cell cycle distribution. Flow cytometry analysis of ASC/TMS1-transfected 786–0 and A498 revealed a significant decrease in the number of cells in the S phase compared with controls Figure , conferrTo investigate the effect of ASC/TMS1 on RCC cell migration, the monolayer wound-healing assay was performed. A significant delay in the closure of the wound gaps in 786–0 cells transfected with ASC/TMS1 as compared with cells transfected with empty vector was observed at both 24 and 36 h Figure . For theTo determine whether the ASC/TMS1-mediated growth inhibition was the result of apoptosis, we performed an apoptosis assay using Annexin V:PE (BD Biosciences) and 7-amino-actinomycin (BD Biosciences) double staining. Ectopic expression of ASC/TMS1 resulted in a significant increase in apoptotic cells as compared with vector control in 786–0 cells Figure . In addiTo further confirm the role of ASC/TMS1 in cell growth and apoptosis, the effect of ASC/TMS1 was investigated through knockdown ASC/TMS1 with small interfering RNA (siRNA) in normal human embryonic kidney cell line HEK293 and RCC cell line Caki-2. Reduced expression of ASC/TMS1 was evidenced by western blot Figures  and 6C. in vitro, we tested whether ASC/TMS1 could alter growth of RCC cells in vivo. The tumor growth of 786–0 stably transfected with ASC/TMS1 or empty vector in SCID mice was shown in Figure P < 0.0001; Figure in vivo.In light of the observed anti-proliferative and pro-apoptotic effects of ASC/TMS1 on the cell lines As RCC are resistance to radiation and chemotherapy, therefore we explored whether restoration of pro-apoptotic gene ASC/TMS1 might sensitizes human renal cancer cells to DNA damaging agents. As a result, up-regulation of ASC/TMS1 by pre-treatment with 5-Aza or recombinant expression in human renal cancer 786–0 cells, in which ASC/TMS1 is silenced by aberrant methylation, potentiated cell death mediated by DNA damaging agent etoposide and doxorubicin Figure  and 8B.To verify the above-mentioned observation that ASC/TMS1 expression sensitizes renal cancer cells to DNA damaging agents, we knockdowned ASC/TMS1 expression in human renal cancer Caki-2 cells, in which ASC/TMS1 is highly expressed. ASC/TMS1 expression in Caki-2 cells was markedly decreased by transfection with a siRNA specific to ASC/TMS1 Figure . As it hIn this study we found that ASC/TMS1 mRNA is widely expressed in normal adult renal tissues and human embryonic kidney cell line HEK293, whereas frequently silenced or downregulated in RCC cell lines and primary RCC tumors. This finding was further confirmed by immunohistochemistry, where a remarkable loss of ASC/TMS1 immunostaining in most studied primary renal tumors was contrasted by a strong, diffuse cytoplasmic and nucleus staining in adjacent non-malignant renal tissues, as has been observed in other normal tissues . Regrettin vitro, we studied tumor suppressive effect of ASC/TMS1 against renal tumor formation in vivo. Our result showed that the tumor growth was significantly retarded in SCID mice inoculated with 786–0/ASC/TMS1 compared with those inoculated with 786–0/empty vector subcutaneously. Taken together, the consistent results observed in vitro and in vivo indicated for the first time that ASC/TMS1 functions as a tumor suppressor in renal carcinogenesis. This understanding of the anti-tumor effect of ASC/TMS1 in RCC suggests that restoration of the function of ASC/TMS1 could halt or reverse the abnormalities, thus having a potential therapeutic effect.ASC/TMS1 gene inactivation in RCC cell lines and tumor tissues, but expression in normal renal tissues and human embryonic kidney cell line suggested that ASC/TMS1 could be a potential tumor suppressor and its downregulation could have functional role in the development of renal cancer. Therefore, the biological function of ASC/TMS1 in RCC was further investigated by gain- and loss-ASC/TMS1 function assays. Ectopic expression of ASC/TMS1 in the silenced A498 and 786–0 cells significantly inhibited cell proliferation and arrested cell cycle at G0/G1 phase. Concomitantly, ectopic expression of ASC/TMS1 significantly decreased cell migration and cell invasion ability in RCC cell lines Figure  and 5B. Given these data apparently suggesting that ASC/TMS1 could be a growth inhibitor in renal cancer, thus a better understanding of the mechanism of the action of ASC/TMS1 is required. ASC/TMS1 has been reported as a pro-apoptotic gene which encodes a 22 kDa protein containing a COOH-terminal caspase recruitment domain (CARD) that promotes apoptosis and a NH3-terminal Pyrin domain (PYD) involved with the nuclear factor-κB pathway. Both PYD and CARD domains are members of the death domain superfamily, which mediates assembly of complexes that mediate apoptotic and inflammatory signaling pathways . The celThese reports indicated the critical role of ASC/TMS1 in apoptosis. We thus analyzed the effects of ASC/TMS1 on cell apoptosis in two human RCC cell lines A498 and 786–0. Increased apoptosis was revealed in ASC/TMS1-reexpressed cells by FACScan analysis Figure . The indIt has been reported that p53 is a pro-apoptotic gene which can regulates both the intrinsic mitochondrial and extrinsic death receptor apoptotic pathways. Recently, Drexler et al.  demonstrBased on the western blot analyses, the cell cycle arrest in G0/G1 phase caused by ASC/TMS1 was mostly attributed to the upregulation of p21 and downregulation of cyclin D1 Figure  and 4E. Cell migration and invasion are critical events during the cancer progression to metastasis. Re-expression of ASC/TMS1 inhibited the cell migration, as demonstrated by the wound healing assay Figure . This efp = 0.005), whereas no significant correlation was found between its methylation and gender, age, tumor location, TNM stage and histological type. Previous studies [To investigate the clinical application of ASC/TMS1 in renal tumorigenesis, we examined the promoter methylation of ASC/TMS1 by methylations pecific PCR in 202 primary RCCs and 25 normal controls. ASC/TMS1 gene promoter was found to be commonly methylated in RCCs  as compared with normal controls . Recognizing the tumor suppressor effect of ASC/TMS1, the inactivation of this gene by promoter hypermethylation would favor tumor progression and a worse outcome. Therefore, the clinical significance of ASC/TMS1 promoter hypermethylation and its associations with patient outcome were evaluated in 202 primary RCC patients. As shown in Table  studies  have sho studies . Our stuRadical nephrectomy is effective to cure local and early RCCs, however, 30% of patients develop metastases after surgery due to its resistance to radiation and chemotherapy. The molecular mechanisms responsible for chemotherapy-resistance in RCC remain unclear. Current studies have reported that in cancer cells, defects in the apoptotic pathway contribute to resistance to chemotherapy . EpigeneAs a result, up-regulation of ASC/TMS1 by pre-treatment with 5-Aza or recombinant expression in human renal cancer 786–0 cells, in which ASC/TMS1 is silenced by aberrant methylation, potentiated cell death mediated by DNA damaging agent Figure  and 8B. In conclusion, we have identified a novel functional tumor suppressor gene ASC/TMS1 inactivated by promoter hypermethylation in RCC, with important functions in suppressing cell proliferation, inducing apoptosis and inhibiting cell invasion. ASC/TMS1 plays an important role in activation of p53 and its downstream targets including p21, Cyclin D1, PCNA, active form of caspase-8, caspase-9, and poly (ADP-ribose) polymerase (PARP). ASC/TMS1 tumor specific methylation may serve as a biomarker for early detection and prognosis prediction of RCC, especially when tumor suppressor gene (TSG) hypermethylation can be detected in patients' serum and urine samples, just like RASSF1A, tissue inhibitor of metalloproteinase-3 and CDH1 methylation . FurtherAll human primary RCCs (202 cases) and adjacent nonmalignant renal tissues were obtained from the urology department of Peking University First Hospital in Beijing from September 2012 to May 2014, with patients'consent according to the university policy. All cases were collected from primary surgical resection with no prior history of RCC and adjuvant therapy. Specimens were snap frozen in liquid nitrogen and subsequently stored at −80°C for molecular analyses. Pathological diagnosis was done and confirmed at the pathology department, Institute of Urology, Peking University First Hospital. The histopathology of tumors was classified by 2009 AJCC TNM stage and Fuhrman nuclear grade.Six RCC cell lines  obtained from American Type Culture Collection and the normal human embryonic kidney cell line HEK293, which serves as a “normal” control for RCC, were routinely maintained in RPMI 1640 or DMEM medium supplemented with 10% fetal bovine serum  in a humidified incubator at 37°C with an atmosphere of 5% CO2.The expression vector (PEX-2–ASC/TMS1) encoding the full length open reading frame of human ASC/TMS1 gene and the control empty vector PEX-2 were constructed by Su Zhou GenePharma, China. Sequence corresponding to the open reading frame of ASC/TMS1 was amplified by PCR and verified by DNA sequencing. The siRNA specific for ASC/TMS1 and its control siRNA were purchased from OriGene .For 5-Aza-2′-deoxycytidine  and Trichostatin A (TSA)  treatment, cell lines were grown in a 6-well plate and treated with 10 μM 5-Aza-2′-deoxycytidine for 72 hours and subsequently with or without 100 nM trichostatin A for 24 hours, as described previously . ControlTotal RNA was extracted from RCC cell lines using Trizol reagent . For semiquantitative RT-PCR, ASC/TMS1 gene was amplified using AmpliTaq Gold DNA polymerase  as reported previously. Real-time PCR was performed using GoTaq(R) qPCR Master Mix  according to the manufacturer's protocol on 7500 Fast Real-Time PCR System (ABI). Primer sequences and PCR conditions are listed in Table Immunohistochemical analysis of ASC/TMS1 protein expression was performed on 67 formalin-fixed, paraffin-embedded tumor tissues and paired adjacent nontumor tissues. Briefly, the sections (4-μm thickness) were deparaffinized in xylene and rehydrated by transfer through graded concentrations of ethanol to distilled water, and endogenous peroxidase activity was blocked by incubation with 3% H2O2 for 15 minutes at room temperature. Then, sections were submitted to antigen retrieval in a microwave  for 10 minutes, naturally refrigerated to room temperature. Blocking was performed with 10% goat serum for 30 minutes at room temperature. All sections were incubated with rabbit anti-ASC/TMS1 monoclonal antibody , overnight at 4°C, and then incubated with goat anti-rabbit secondary antibody for 30 min at room temperature. After rinsing three times in PBS for 5 min each, the sections were incubated with DAB for 2 min, counterstained with hematoxylin for 3 min, dehydrated with gradient alcohol and transparentized with dimethylbenzene. Immunohistochemical expression of ASC/TMS1 was examined via light microscopy. Positivity was ascertained based on the presence and intensity of brown granules. All slides were reviewed independently by 2 pathologists who were blinded to each other's readings. Tissues were graded on the following scale: 0, negative; 1, weakly positive; 2, moderately positive; 3, strongly positive.To check ASC/TMS1 protein expression after 5-Aza treatment, we performed immunofluorescence staining against ASC/TMS1 in 786–0 cells. Cells grown on glass coverslips were fixed with freshly prepared 4% paraformaldehyde for 15 min, followed by permeabilization with 0.1% Triton X-100 for 5 min on ice. Then, after washing with PBS, the cells were blocked with goat serum for 30 min. For immunofluorescence, Cells were incubated with primary antibody against ASC/TMS overnight at 4°C followed by secondary goat anti-rabbit antibody conjugated with FITC  at room temperature for 45 min. The nuclei were counterstained with DAPI  and viewed under a fluorescence microscope.The bisulfite modification of purified genomic DNA was performed using an EpiTect Bisulfite Kit  following the manufacturer's instructions. MSP primers were examined previously for not amplifying any unbisulfited DNA and MSP products of several RCC cell lines were confirmed by direct sequencing, indicating that our MSP system was specific. For bisulfite genomic sequencing, bisulfite-treated DNA was amplified with primers specific for a fragment of the ASC/TMS1 promoter CpG islands that contained 53 CpG sites. The PCR products were subcloned into the pEasy-T5 vector  and 5–8 colonies were randomly chosen and sequenced. MSP, BGS primer sequences and PCR conditions are listed in Table Cell viability was measured using the Cell Counting Kit-8  according to the instructions of the manufacturer. Briefly, A498 and 786–0 cells (2 × per well) were seeded in 96-well plates and transient transfected with PEX-2-ASC/TMS1 vector or PEX-2 empty vector. HEK293 (5 × per well) was seeded in 96-well plates and transient transfected with ASC/TMS1 siRNA or control siRNA. After transfection, at 24-hour intervals for 6 days, 10 μl of CCK-8 solution was added to each well of the plate, and the plate was incubated at 37°C for 1 hour. The absorbance was measured at 450 nm as an indicator of cell viability. All experiments were independently repeated at least 3 times.Cells were plated in a 12-well plate and transfected with expression plasmid PEX-2-ASC/TMS1 or the empty vector PEX-2 (0.8 ug each) using Lipofectamine 2000. At 48 hours after transfection cells were collected and replated in a 6-well plate and selected for 2 weeks with G418 (0.4 mg/ml). Surviving colonies (50 or greater cells per colony) were counted after staining with crystal violet. All experiments were independently repeated at least 3 times.Cell migration was assessed using a scratch wound assay. Briefly, PEX-2-ASC/TMS1 or empty vector stably transfected 786–0 cells were cultured in six-well plates. When the cells grew up to 80–90% confluence, a wound was made by dragging a plastic pipette tip across the cell surface. The phase contrast images of the wounds were recorded at 37°C for incubations of 0, 12, 24, 36 hours, and 3 separate experiments were performed.Cell invasion assays were performed using 24-well transwells , coated with Matrigel . RCC cells were starved overnight in serum-free medium, trypsinized, and washed 3 times in DMEM containing 1% FBS. A total of 1 × 105 cells was then suspended in 500 μl DMEM containing 1% FBS and added to the upper chamber, while 750 μl DMEM containing 10% FBS was placed in the lower chamber. For the control, medium containing 1% FBS was added to the lower chamber. After 24 hours of incubation, Matrigel and cells remaining in the upper chamber were removed by cotton swabs. Cells on the lower surface of the membrane were fixed in 4% paraformaldehyde and stained with 0.5% crystal violet. Cells in at least 6 random microscopic fields  were counted and photographed. All experiments were repeated 3 times.The stably transfected 786–0 cells with PEX-2-ASC/TMS1-expressing or PEX-2- and A498 ASC/TMS1 empty vector were fixed in 70% ethanol and stained with 50 mg/ml propidium iodide . The cells were then sorted by FACSCalibur  and cell cycle profiles were analyzed by ModFit 3.0 software . Apoptosis was assessed using the dual staining with Annexin V:PE (BD Biosciences) and 7-amino-actinomycin (BD Biosciences). Briefly, PEX-2-ASC/TMS1 or empty vector-transfected cells were harvested at 48 h post transfection. Annexin V:PE and 7-amino-actinomycin were added to the cellular suspension according to the manufacturer's instructions and was analyzed by flow cytometry . Both early and late apoptotic cells were counted for relative apoptotic changes. All the experiments were performed at least three times.Total protein was extracted with RIPA (Radio-Immunoprecipitation Assay) lysis buffer, supplemented with 1mM Na3VO4, 1mM NaF, 1X protease inhibitor and 1mMPMSF before use. Equal amounts of proteins were separated by 10–12% SDS-PAGE gel and then transferred onto nitrocellulose membranes. After blocking the membranes with 5% fat free milk in TBST for 1 h at room temperature, the membranes were incubated with specific primary antibodies overnight at 4°C. After washing, the membrane was incubated with HRP-conjugated secondary antibodies for 1 h at room temperature and then washed with TBST. The antibody complexes in the immunoblots were detected by chemiluminescence using an HRP substrate  and visualized using a G:BOX Chemi Gel Documentation System .Cells were transfected with a control non-targeting siRNA or with an ASC/TMS1-targeting siRNA (50 nM) using Lipofectamine 2000 according to the manufacturer's instructions. For DNA damaging agent treatment, after 48 h of transfection, Caki-2 cells were treated with etoposide (50 uM) or doxorubicin (1 uM) and analyzed by Western blot for the indicated antibodies.6 cells in 0.2 ml phosphate-buffered saline) stably transfected with PEX-2-ASC/TMS1 vector or PEX-2 empty vector were injected subcutaneously into the dorsal right flank of 4-week-old male SCID mice, separately. Tumor diameter was measured every 3 days for 18 days. Tumor volume (mm3) was estimated by measuring the longest and shortest diameter of the tumor and calculating as follows: volume = (shortest diameter)2 × (longest diameter) × 0.55 as described previously [786–0 cells  of n observations. The significance of differences between two groups was determined using a two-tailed


Biliary atresia and choledochal cyst have the similar clinical presentation in infants. Herein, we report a case that presented with prolonged hyperbilirubinemia and abdominal distension and diagnosed as choledochal cyst. At surgery, in addition to a large choledochal cyst, biliary atresia was also encountered. A 22-day-old female infant was admitted due to prolonged jaundice. She was icteric from the fourth day of life along with acholic stools and abdominal distension. Direct hyperbilirubinemia was detected in blood tests. Her total bilirubin and direct bilirubin were 19mg/dl and 10.3mg/dl respectively. Clotting profile was normal. Liver function tests (LFTs) were AST (84IU/L), ALT (81IU/L), and Alkaline phosphate (2024 IU/L). The abdominal sonography showed a choledochal cyst (56x43x46mm) with few echogenic areas in it. Wall thickness and echogenicity of gallbladder as well as intra hepatic ducts were normal. Abdominal CT scan confirmed the existence of choledochal cyst . The patient was operated after optimization. Operative findings included an extra hepatic choledochal cyst (type I) which was ending blind on either sides. Porta-hepatis showed type III biliary atresia. The choledochal cyst and gallbladder had no bile but mucinous secretions which were aspirated. The gallbladder and choledochal cyst were resected; the porta-hepatis dissected and Roux-en-y porto-enterostomy was performed. The wedge biopsies from porta-hepatis showed biliary atresia as donated by diffused feathery degeneration, cholestasis, pseudoacinar transformation, focal giant cell, bile duct proliferation, and fibrotic tissue. The histopathology of choledochal cyst showed a biliary epithelial lining and that of gallbladder showed congestion and near total obliteration. Postoperatively, the jaundice improved and the patient passed pigmented stool. Patient was allowed orally and discharged on short course of prednisolone, fat soluble vitamins and prophylactic antibiotic. At present, the infant is 10- months old and thriving well. The last total bilirubin is less than 1.5mg/dl. The infantile choledochal cyst presents with obstructive jaundice, acholic stools, and hepatomegaly resembling biliary atresia and even may lead to advanced liver fibrosis.[1] In patients with biliary atresia, gallbladder is either not visualized or atretic.[2] Ultrasonographic triangular cord sign may be seen in patients with biliary atresia. Although ultrasonography may help differentiate between both but occasionally the occurrence of both cannot be picked on it. DISIDA scan may help but as in the present case there was a cyst with patent looking gallbladder, we did not perform it. Few similar cases have been reported in literature where patient was preoperatively labelled as choledochal cyst but operation revealed biliary atresia as well. In differentiating simple choledochal cyst from choledochal cyst associated with biliary atresia, the accuracy of abdominal sonography will increase if the diameter of the extrahepatic cyst dilatation is more than 1.5 cm without an atretic gallbladder. We stress that in infantile choledochal cyst, DISIDA or HIDA scan should be added to ultrasound or CT scan that may show absent excretion of radiolabelled contrast into the duodenum thus giving a suspicion of associated biliary atresia. MRCP however is superior tool in diagnosing this concurrence.[5] Source of Support: NilConflict of Interest: None


A1CF) encoding the complementation factor for the APOB mRNA-editing enzyme is associated with urate levels. However, the relationship of apo B and VLDL with gout and hyperuricaemia (HU) is still unclear. Therefore, we tested the association of VLDL and apo B with HU and with gout compared to HU.Gout results from an innate immune response to monosodium urate (MSU) crystals deposited in joints. Increased very low-density lipoprotein (VLDL) has been associated with gout. The apolipoprotein B (apo B), which is present on VLDL, regulates neutrophil response to MSU crystals and has been positively associated with gout. Furthermore, the gene  and Māori and Pacific Island (Polynesian) (n = 90) male gout case and control sample sets were divided into normouricaemia (NU), asymptomatic HU and gout groups. Size exclusion chromatography and enzyme-linked immunosorbant assay was used to measure VLDL and apo B. Multivariate logistic regression was used to assess the risk of gout and HU per unit change in VLDL and apo B.P = 1.8 × 10-6 and 1 × 10-3, respectively), but only compared to NU in Polynesians (P = 0.023). This increase was driven by increased number of VLDL particles in the European participants and by the Tg-enrichment of existing VLDL particles in the Polynesian participants. Each mmol/L increase in VLDL Tg was significantly associated with gout in the presence of HU in Europeans, with a similar trend in Polynesians . Each μmol/L increase in total apo B trended towards decreased risk of HU  and, conversely, with increased risk of gout compared to HU .Increased levels of VLDL triglycerides (Tg) were observed in the gout sample set compared to NU and HU in Europeans (Increased VLDL Tg is associated with the risk of gout compared to HU. A genetic approach should be taken to investigate the possibility for causality of VLDL in gout. Apolipoprotein B may have pleiotropic effects in determining HU and gout. The genetic and environmental causal factors in determining hyperuricaemia (HU) are becoming better understood ,2. Howevet al.  demonstret al. ,7. Elevaet al. . Intereso B mRNA ,10.Collectively the observations outlined above implicate VLDL Tg and/or apo B in gout. However, the relationship between VLDL-Tg, apo B and HU and gout is unclear. We hypothesized that if increased VLDL Tg plays a role in regulation of the innate immune response to MSU crystals in gout then this would be consistent with association of VLDL Tg with gout compared to HU. We also tested for association of circulating levels of apo B with HU and gout compared to HU.The 180 New Zealand (NZ) male participants consisted of gout cases, defined by the American Rheumatology Association preliminary classification criteria , and conFast protein liquid chromatography (FPLC) was used to fractionate lipoproteins from frozen non-fasting serum samples. Tg and cholesterol assays were done on the FPLC fractions to identify lipoprotein-containing fractions. Dilutions of Precipath L  were used to make a standard curve and all samples were tested in triplicate. A total of 100 μL of each fraction was incubated with 100 μL of Tg glycerol-3-phosphate oxidase-phenol and aminophenazone (GPO-PAP) reagent (Roche/Hitachi) or cholesterol oxidase (CHOD)-PAP reagent (Roche) for 15 to 20 minutes at 37°C in a 96-well plate . Non-fasting serum samples were studied because participants were convenience sampled from health care and community settings.2) = 175 × ((SCr × 0.0113)-1.154 × (Age-0.203).An enzyme-linked immunosorbant assay (ELISA) was used to measure apo B in serum and FPLC fractions as previously described . Cfas Lit test or chi-square test via the Intercooled STATA™ software version 8.0 . Multivariate-adjusted linear and logistic regression analyses were used to assess the association of VLDL and other lipid variables with phenotype. A threshold of P <0.05 was used to indicate statistical significance.Due to the skewed distribution of lipid-related phenotypes, all lipid, lipoprotein and apolipoprotein values were expressed as median and interquartile range (25th to 75th percentile) and pairwise comparisons were done using the Wilcoxon-Mann-Whitney test or Student P = 1.84 × 10-6 and 0.001, respectively) whereas the difference was significant between NU and gout in Polynesians (Table P = 0.023). FPLC is a standard methodology used to separate lipoproteins by size [P = 0.0041 to 1.23 × 10-06), with the ratio increasing from NU to HU to gout. However, in the Polynesian groups, this ratio was not significantly different between NU and HU controls (P = 0.44) but was significantly increased in gout compared to NU and HU (P = 0.0071 and 0.0011 respectively).Traces of Tg concentrations in FPLC fractionated lipoproteins Figure  indicate by size . The areP = 0.018 and 4.5x10-5, respectively) and in gout compared to HU (P = 0.041). In the Polynesian group the corresponding value for VLDL apo B concentration was not significantly different between gout and the other subgroups, although it was higher in gout. There was a significant increase in the VLDL apo B to total apo B ratio in HU and gout compared to NU in Europeans and between NU and HU in Polynesians . Collectively these results suggested an increased number of VLDL particles in HU particularly in Europeans, with an increase in Tg loading of these particles evidenced in gout in Polynesian participants.The VLDL apo B concentration was significantly higher in HU and gout cases in Europeans compared to NU (Table ns Table . There wP = 0.011) and a trend towards increased risk in Polynesians (odds ratio (OR) = 2.84; P = 0.069), with significantly increased risk in the combined ancestral groups . These results were robust to exclusion of gout cases taking fenofibrates or statins (Table P = 0.014) but not Polynesians . There was no significant risk in gout associated with total serum Tg.We tested the risk of gout compared to NU and HU for three Tg phenotypes  (Table A1CF gene [P = 0.044) but not in Europeans  with trending evidence for a protective role in HU in the combined sample sets . In contrast, each 100 nmol/L increase in VLDL apo B was not associated with an increased risk of HU in Europeans  or Polynesians  although there was evidence for increased risk of HU in the combined sample sets . There was, however, by linear regression analysis no association of apo B with urate levels in the combined NU and HU groups (Table P = 0.004 per μmol/L unit change) although there was no evidence for association of VLDL apo B . Thus apo B negatively correlated with HU, but positively correlated with gout in the presence of HU.We next investigated apo B. It was notable that total apo B was significantly lower in HU than in NU in both ancestral groups .Given the organised chronic inflammatory nature of the tophus, with inflammatory factors that may promote resolution of gout attacks  and thusP = 0.011), although there was a trend towards increased risk in Polynesian participants . We also associated reduced total apo B with HU and increased total apo B with gout compared to HU. Given this is a cross-sectional observational study, these results cannot ascribe causality to VLDL Tg and/or apo B in HU and in gout compared to HU. However the data, when considered with the previous literature, support further investigation of the role of VLDL Tg and/or apo B in HU and gout.We observed increased levels of VLDL Tg in gout compared to asymptomatic HU. This increase was suspected to be driven by overproduction of VLDL particles in HU particularly in Europeans  could be involved in gout in the presence of HU.Reduced total apo B was associated with HU whereas increased total apo B was associated with gout compared to HU. These observations suggest pleiotropic effects of apo B in gout. Previously, serum urate control in Europeans has been associated with genetic variation in the 1CF gene , which e1CF gene . While ihin A1CF  suggestiThe fibric acid derivative fenofibrate is used in hypertriglyceridaemia to reduce VLDL, LDL and total triglycerides. It activates peroxisome proliferator-activated receptor alpha, which in turns activates lipoprotein lipase to reduce circulating Tg-rich particles . In primOne possible limitation of our study relates to the non-fasting status of the participants. This would have introduced additional variability into the Tg but not the apo B data - in a study of healthy subjects at four and eight hours postprandially, total apo B did not differ whereas total Tg levels increased when compared to fasting . It is iGCKR, MLXIPL, A1CF) [Consideration of genetic data does support the hypothesis that aspects of Tg metabolism play a causal role in gout. For example, there are loci associated with control of serum urate levels that contain genes that are also associated with serum Tg levels   and goutL, A1CF) ,10. A MeL, A1CF) . These gWe report association of reduced total apo B with HU, but increased apo B and VLDL Tg with gout compared to HU. These data support the hypothesis that VLDL metabolism plays a role in gout, with apo B having pleiotropic effects.


Neonatologists generally recognize that late preterm infants face more problems in the immediate newborn period compared with their full-term counterparts. ,2. This The most widely studied metric of health care utilization in late preterm infants is short-term readmission (the first two weeks) after birth hospitalization. Escobar  found thEscobar examinedThe late preterm infant is particularly responsive to the benefits but vulnerable to the risks of early discharge home. Longer length of stay before discharge is protective against readmission but it is not reasonable to prolong the birth hospitalization of newborns who meet criteria for discharge. More effort needs to be placed to reduce the risk of jaundice and feeding problems in these patients: to avoid mother and infant separation during birth hospitalization, to arrange a follow-up appointment within 48 hours of discharge, to promote and support lactation before and after discharge.The author has no conflict of interest to declare.


Birth asphyxia is an insult to the fetus or newborn due to failure to breath or breathing poorly, leads to decrease oxygen perfusion to various organs. According to WHO, 4 million neonatal deaths occurred each year due to birth asphyxia. Our goal was to evaluate antepartum, intrapartum, and fetal risk factors of birth asphyxia.It was a Retrospective Case control study, conducted at Neonatal Intensive Care Unit of pediatric ward  and in Gynecology wards  of Civil Hospital Karachi, Dow University of Health Sciences. Study was conducted from January 2011-November 2012. Neonates diagnosed with birth asphyxia were considered as “cases” while neonates born either with normal vaginal delivery or by cesarean section having no abnormality were considered as “control”. Demographics of both the mother and neonate were noted and Questions regarding possible risk factors were asked from mother. Ethical issues were confirmed from Institutional review board of Civil Hospital Karachi, Dow University of Health Sciences. All data was entered and analyzed through SPSS 19.Out of total 240 neonates, 123 were “cases” and 117 were “control”. Mean maternal age in “case” group was 24.22 ± 3.38 while maternal age of control group was 24.30 ± 4.04. Significant antepartum risk factors were maternal age of 20–25 (OR 0.30 CI 95% 0.07-1.21), booking status (OR 0.20 CI 95% 0.11-0.37), pre-eclampsia (OR 0.94 CI 95% 0.90-0.98) and primigravidity (OR 2.64 CI 95% 1.56-4.46). Significant Intrapartum risk factors were breech presentation (OR 2.96 CI 95% 1.25-7.02), home delivery (OR 16.16 CI 95% 3.74-69.75) and maternal fever (OR 10.01 CI95% 3.78-26.52). Significant Fetal risk factors were resuscitation of child (OR 23 CI 95% 31.27-1720.74), pre-term babies(OR 0.34 CI 95% 0.19-0.58), fetal distress (OR 0.01 CI 95% 0.00-0.11) and baby weight (OR 0.13 CI 95% 0.05-0.32).Measures should be taken to prevent neonatal mortality with great emphasis on skilled attendance at birth and appropriate care of preterm and low birth weight neonates. Birth asphyxia, although the correct definition is imprecise, is an insult to the fetus or newborn due to failure to breath or breathing poorly leading to decrease oxygen perfusion to various organs. According to WHO, 4 millions deaths yearly occurred due to birth asphyxia, representing 38% of all deaths of children under 5 years of age. In low-income countries 23% of all neonatal deaths occurred due to birth asphyxia . AccordiAsphyxia is a condition that occur when there is an impairment of blood-gas exchange, resulting in hypoxemia (lack of oxygen) and hypercapnia (accumulation of carbon dioxide). The combination of the decrease in oxygen supply (hypoxia) and blood supply (ischemia) results in a cascade of biochemical changes inside the body, whose events lead to neuronal cell death and brain damage. Continuous asphyxia will also lead to multiple organ systems dysfunction. Birth asphyxia is a serious clinical problem worldwide and contributes greatly to neonatal mortality and morbidity .Causes of perinatal birth asphyxia may be maternal or fetal. Those who survive asphyxia at birth may have chance to develop neurological complications including epilepsy, cerebral palsy and developmental delay . Risk faAccording to a study in Pakistan, birth asphyxia contributed to 16.52% of hospital admissions and is responsible for 21% of infant deaths .However, Pakistan being the developing country, experiences a significant morbidity due to birth asphyxia and its sequel of complications. In Pakistan there is not much data available on the risk factors of birth asphyxia, hence studies are required to evaluate the risk factors of birth asphyxia so that interventions can be done to educate and guide people about the risk factors and management strategies. Our main goal was to evaluate the antepartum, intrapartum and fetal risk factors of birth asphyxia.It was a Retrospective case control study conducted at the neonatal intensive care unit of pediatric ward  and at Gynecology ward  of Civil Hospital Karachi, one of the largest public sector, tertiary care hospitals in Karachi. Study was done from January 2011-November 2012.Subjects were divided into cases and control. Neonates diagnosed with birth asphyxia were considered as “cases” while neonates born from normal vaginal delivery or by cesarean section with no abnormality were considered as “control”. Sample size was determined with the help of Open EPI calculator.Gestational age after viability.Severe Asphyxia: Apgar score at 1 min is 0–3.Diagnostic criteria of Birth Asphyxia:c)Absence of major congenital malformation.d)Multi organs failure in the first 72 hours or convulsion in the first 24 hours of life.e)History of delayed cry.Mild-Moderate Asphyxia: Apgar score at 1 min is 4–7Birth weight less than 1000 g pre-maturely.Opium or Anesthesia related Low APGAR score.Babies with lethal anomalies like hydrops, cyanotic congenital heart defects, congenital or chromosomal anomalies and congenital infections.Exclusion Criteria were:Apgar scoring consists of five physical sign: heart rate, respiratory effort, reflex irritability, muscle tone and color. Labor was considered prolonged when it exceed 12 hours in primigravida or 8 hours in multipara . Fetal dIntrapartum: Event occurs during labor and delivery.Antepartum: Event occurring before childbirth, with reference to the mother.Maternal characteristic were obtained during interview of mothers, using pre-structured Performa.Before entry into the study, subjects were informed about the aims, methods and anticipated benefits. Subjects will be informed that their participation is voluntary. They will be informed that choosing not to participate will not affect their care. After giving sufficient information written consent was obtained and confidentiality of data was maintained.Performa was filled on each case. Questionnaire comprises of 24 questions. 1st portion comprise of demographic data of neonate and mother. Q1-Q7 comprises of questions regarding age of mother, type of case (booked or not), any illness during pregnancy, drug and smoke history, type of pregnancy, history of diabetes, anemia and any previous history of birth asphyxia. Second section comprise of history of current pregnancy. It consist of questions from Q8-Q18, these were regarding any fetal condition diagnosed during pregnancy, presentation of fetus, mode of delivery, type of assisted vaginal delivery, anesthesia received during C-section, history of prolong labor, place of delivery, any emergency complication face by mother and history of sedation. Last section was regarding neonatal characteristic. It compromise of questions from 19–24, which consist of gestational age at birth, history of cry and seizure, about resuscitation, mode of resuscitation and birth weight. Information about the date and time of birth, gender, birth weight and result of Apgar score at 1, 5 and 20 minutes has been noted in questionnaire.Study was approved from the institutional review board of Civil Hospital Karachi, Dow University of Health Sciences.All data was entered and analyzed through SPSS 19. Mean and standard deviation were used for continuous data while frequency and percentage were calculated for categorical data. Risk factors for birth asphyxia were grouped into antepartum, intrapartum, and fetal variables. Association between birth asphyxia and risk factors was determined by Binary logistic regression. The percentage of risk factors associated with birth asphyxia at different intensity levels was estimated by odds ratio with the confidence interval CI of 95%. Threshold of significance was set at 0.05During the period of two years (January 2011-November 2012), 240 cases fulfilled the inclusion criteria. Out of these 240, 123 were of birth asphyxia (cases) and rest of 117 were normal (control). Male neonates were 147 (61.3%) and females were 93 (38.8%). Mean maternal age in asphyxia group was 24.22 ± 3.38 while mean maternal age of control group was 24.30 ± 4.04.Mothers at age of 20–25 were at higher risk of developing Birth asphyxia as compare to younger or elder mothers (<20 or >25) . Risk increased significantly with decline in booking status of mother . Pre-eclampsia was associated significantly with increase risk of birth asphyxia . Gestational diabetes was associated with development of birth asphyxia .Maternal hypertension (p = 0.46), anemia p = 0.10) and diabetes mellitus (p = 0.93) were not related to an increase risk of birth asphyxia  and diuretics  were associated significantly with increase risk of developing AsphyxiaInfants of primiparous women carried a higher risk for birth asphyxia 70 (56.9%) as compared to normal 39 (33.3%) . Mother of neonates who developed birth asphyxia was mostly belonging to low socio economic group 62 (50.4%) and they have no prior history of abortion 102 (82.9%). Mothers having prior history of asphyxic child have more prone to birth asphyxia again as compare to control (OR 3.92 CI 95% 1.26-12.19)  as compare to control 2 (1.7%) . Deliveries by midwives were significantly associated with increase risk of birth asphyxia . Normal vaginal delivery was frequent in both asphyxia 77 (62.6%) and control group 68 (58.1%) and there were also no association to be found with type of anesthesia received by mother. Breech presentation of fetus was significantly associated with increased risk of developing birth asphyxia  , umbilical cord complication like prolapsed umbilical cord , cephalopelvic disproportion , prolonged labor  and Spontaneous premature rupture of membrane  were significantly associated with increase risk of developing birth asphyxia  as compared to the infant with weight of 2.5- to >3.5 kg. Prematurity carried a substantially higher risk of developing birth asphyxia, with gestational age of 34 to 37 weeks, increasing the risk of asphyxia by a factor of 0.34 (CI 95% 0.19-0.58). Fetal conditions like oligohydromnios , pre-mature delivery  and fetal distress  were significantly associated with increment in the risk of developing birth asphyxia. Intra uterine mecoinum release was significantly present in asphyxia cases 24 (19.5%) (p = <0.001). Amniotic fluid surfactant test were found negative only in asphyxia cases 9 (7.37%)  and primigravidity has been one of the main risk factors of developing birth asphyxia as mentioned in previous studies ,11-13. PTo reduce the risk factors of causing birth asphyxia in low income and developing countries is not an easy task due to certain reasons. One of them was the delivery conducted by untrained traditional midwives as indicated in previous reports also ,14. ThisTwo important risk factors mentioned in our study were pre-term delivery and maternal fever, same as mentioned by Lee et al. and Khreisat et al. ,16. TheiRegarding mode of delivery it showed that most of the cases and control were delivered by normal vaginal delivery, this result was very much similar with the findings of two studies conducted in Pakistan, 2012 on same issue ,18.In our study, meconium stained amniotic fluid was found to be present as one of the risk factor, findings were comparable with previous study also . In healBreech presentation exhibited a 2.96 times higher risk of birth asphyxia than other presentations, results were similar to previous studies ,19. It mLow birth weight was one of the major culprits for causing birth asphyxia . A potenHypertension and anemia were not emerged as a risk factors for birth asphyxia but they found more common in cases as compare to control, same as observed in past studies ,20. HypeIn our study, socioeconomic factors were associated non-significantly with risk of birth asphyxia same as indicated in past study . Pre-eclIn our study, usage of adrenergic drugs and diuretics were found to be the significant reisk factors of birth asphyxia which was not mentioned in any prior study on this subject.This study was hospital based and addressed a common problem of our community Majority of study population as not being delivered under care of trained professionals primarily at home and private clinics also reflects another important issue pertaining to mortality and morbidity.The strength of our study lies in its case control design. Previous studies of Pakistan have targeted few and selected risk factors but we discuss nearly each and every risk factors of birth asphyxia. All attempts were made to ensure that the data collected was reliable and the methods were reproducible. However, our study was not free from limitations.Main limitation was that our study conducted in one tertiary care hospital of Karachi where mostly patients belong to the low and low middle class and data couldn’t predict the overall situation in the country. Second limitation was short sample size of the study which may have limited our ability to detect small differences. Furthermore, convenient sampling was employed, which may have led to selection bias, and hence was not truly representative of the population under study. Third limitation was that, for the evidence of anemia, diabetes and hypertension we mostly relied on the maternal history without consideration of when the events occurred and how long they were or how they were managed.Our research opens the door of large proportion of research which needs on this topic to define more accurately the true burden of birth asphyxia in developing countries, contribution of common risk factors such as maternal malnutrition and infection and outcome of asphyxia, and to intervention strategies that can be applied at scale in developing countries.Sequelae of birth asphyxia vary from no ill effects to multiorgan complications and death. This diversity varies with severity and duration of asphyxia and it may be due to the presence of gaps in the field of research and technology in developing and low income countries.Age of mother, lack of booking status, pre-eclampsia, intake of diuretics and adrenergic drugs were reported as maternal risk factors. Intra partum risk factors are home delivery by midwives, breech presentation, prolapsed umbilical cord, cephalopelvic disproportion and fever. Fetal risk factors are oligohydromnios, meconium stained amniotic fluid, pre-mature delivery, resuscitation of neonate pre-term delivery and low birth weight. Majority of these factors may be manageable by means of good pre-natal care.


To determine frequency of Low Birth Weight (LBW) at term and to determine frequency of various associated risk factors.This cross-sectional study was conducted in Department of Obstetrics and Gynaecology Layari General Hospital and Dow University of Health Sciences Karachi between January 2007 to July 2008. Women with singleton pregnancy of 37 and above completed weeks were identified. Those women who gave birth to babies with less than 2500gm were recruited in the study. In all those patients who gave birth to LBW babies risk factors were assessed. The variables including age, parity, booking status, maternal weight and height, socioeconomic condition, smoking, fetal gender and birth weight were noted on a questionare. The data was analyzed on SPSS Version 15. Frequency of LBW and its associated risk factors were determined.During study period 10.6% patients delivered LBW babies. Antenatal care was not received in 67% patients. Parity was less than three in 87%. In 26% of patients maternal weight was less than 50 kg and in 37% patients with LBW, maternal height was less than five feet. Majority of patients were anaemic (72%) including 20% with haemoglobin < 7 grams.LBW is associated with a group of factors which may be regarded as high risk factors. These include low socio economic status, anaemia, primiparity, short maternal height and less than average weight. More than 70% of these Low Birth Weight (LBW) babies are small due to constitutional and environmental factors. Small for gestational age may be due to pathological reasons when it is called as intra uterine growth restriction.Weight of a baby at term depends on gestational age and rate of fetal growth in uterus. Babies born may be appropriate for gestational age but are small because of preterm delivery. Babies who are small for gestational age may be born preterm or term. A baby is said to be small for gestational age when the gender specific birth weight is below the 10Depending upon birth weight and gestational age, WHOIn developing countries, LBW is a major determinant of perinatal mortality and morbidity. Across the world Neonatal mortality is 20 times more likely for LBW babies as compared to babies heavier than 2.5kg.3An improvement with increment in the birth weight of babies is expected to reduce the occurrence of LBW babies; therefore this study was conducted with the intention to identify risk factors present among mothers who delivered babies with LBW. Identification of factors which carry risk of LBW may help to attempt their correction and counseling when possible. This modification will help in decreasing perinatal mortality and morbidity.This cross sectional study was conducted in Department of Obstetrics and Gynaecology, Dow Medical College & Layari General Hospital Karachi, from January, 2007 to July 2008. The selection criteria were singleton pregnancy with ≥37 completed weeks of gestation. Patients with multiple pregnancy, in-utero death, fetal abnormality, medical disorders like hypertension, diabetes, renal disease and cardio-respiratory disease, antepartum haemorrhage and those who delivered preterm were excluded. All those patients who delivered during the study period and met the inclusion criteria were recruited in the study by designated post graduate present in labor ward. An informed consent was taken from all selected patients. Variables including age, parity, maternal weight and height, family income, haemoblobin (Hb), birth weight, fetal sex were noted on predesigned questionare.After delivery all babies were weighed on scale in labour room after clamping and cutting the umbilical cord, within one hour of birth. Babies were termed as normal weight if birth weight was 2.5 to 4 kg, LBW if less than 2.5 kg and macrosomic if more than 4 kg. Anemia was classified as no anemia if Hb was more than 10 g/dl, mild to moderate if Hb was 7 – 10 g/dl and severe anemia if Hb was less than 7 g/dl.The data were analyzed using SPSS version15. Mean ± SD was calculated for numerical variables like age, parity and gestational age. Frequencies and percentiles were calculated for categorical variables such as birth weight, maternal age, height, weight, parity, booking status, monthly income, anaemia, smoking and fetal gender.The study included 947 singleton live births at term. Birth weight was less than 2.5kg in 100 patients. Mean age of patients was 26 years. Majority of patients (82%) were 20-30 years old . In patiWeight of baby reflects the maternal health and nutritional status of mother before conception and during pregnancy. Growth of the baby in utero is determined by maternal, fetal and placental factors.A baby’s birth weight is related to birth weight of both parents and more strongly through the maternal line.In this study risk factors which are strongly associated with LBW babies are identified as primiparity, low socioeconomic status, anemia, maternal height and weight. In a meta analysis a significant association is noted between abuse and low birth weight.The adverse effects of maternal smoking for human pregnancy are well known. Use of smoking and illicit drugs during pregnancy is associated with pregnancy complication and Low birth weight. Maternal smoking reduces mean birth weight by about 150-200 gm and doubles the risk of LBW associated with restriction of intra uterine growth. Moreover a dose response effect has been described with occurrence of low birth weight increasing with the number of cigarettes smoked per day. The risk of LBW was reported 4.1 times high in women who are addicted to any tobacco product as compared to those who were not exposed to any tobacco. Not only cigarette smoking, tobacco chewing is also a risk factor for LBW.In this cross sectional study the frequency of LBW was found as 10.6%. This is comparable to the figure of 12.7% reported earlier.3Studies have shown that maternal age is an important predictor for the size of the new born. The relation of age and LBW is reported differently by authors. The incidence of LBW was significantly higher in the group of teen age mothers i.e 65.52%.1In the current study primiparity was noted in 54% of patients with LBW. This is consistent with a report from Kozuki who reported nulliparity and para above 3 at risk of having LBW babies.13Modest degree of malnutrition has effect on birth weight of baby. Mother with optimal weight are considered to have babies of average weight. Increased maternal birth weight and pre pregnancy maternal over weight or obesity had a strong co relation with new born high birth weight. A study from New Zealand reports mothers of LBW were shorter and reported lower pre pregnancy weight as compare to mothers of AGA infants.14In this study, 26% of the mothers of low birth babies were less than 50 kg weight and 70% were between 50-54 kg. Zhen Han reported underweight women had an increased risk of LBW babies RR 1.64, 95% CI1.38—1.94.15There is increased risk of LBW of newborn in women of short height.Lack of ANC was reported as significantly associated with LBW. Lin-Lin–Dal suggested number of antenatal visits had a U trend effect on LBW. He reported increasing number of prenatal visits decrease risk of LBW.From India Mumbare reported 62.4% mothers who delivered LBW babies did not receive antenatal care.Severe maternal starvation during pregnancy has a major impact on fetal growth. During winter 1944-1945 Dutch population suffered severe famine and mean maternal caloric intake fell to 450-750 Kcal a day with subsequent decrease of 250 gm in baby’s birth weight. Kayode has reported a high incidence of LBW babies among women living in rural areas with low coverage of safe water supply. This could be because of increased episodes of gastro intestinal infections impairing normal fetal development.20Anaemia is the commonest medical disorder in pregnancy. 56% of pregnant women in developing countries are anaemic. Risk of anaemia in Pakistan ranges from 8-33%. Anaemic mothers tend to deliver small babies. A strong relationship exists between maternal anaemia and LBW babies. Badshah has reported a significantly higher incidence of LBW babies among anaemic as compare to non anaemic mothers.21Lone, et al.25There is an identifiable group of women with high risk for LBW. Low socioeconomic status, anaemia, maternal height and weight are significantly associated with LBW. Correction of anaemia, intake of a balanced diet, utilization of antenatal care is expected to reduce the frequency of LBW babies and decrease perinatal mortality.


Introduction: Recent evidence suggests that preterm birth is a possible risk factor for high blood pressure (BP) in later life. The most widely quoted blood pressure centiles for very low birth weight  infants at corrected term gestation is based on a cohort with mostly late preterm or term infants (Zubrow curves).Objectives: The objective of this study was to determine the clinical utility of the Zubrow curves in diagnosis of hypertension in VLBW infants at their term corrected gestational age (CGA).Patients and Methods: In a case-control study, we compared BP in 75 VLBW infants at 40 weeks CGA (cases) to 69 full term infants admitted to neonatal intensive care unit (NICU) (controls).Results: In spite of having lower weights, VLBW infants compared to term infants  had higher average systolic  and mean BP . Although 41% (31/75) VLBW infants would have met the criteria for hypertension according to Zubrow curves only 4% (3/75) were diagnosed with hypertension.Conclusion: Since Zubrow BP centiles were based on a heterogeneous population of infants including preterm and term infants, new BP centiles based on chronological data from VLBW infants would allow a better definition of hypertension in these infants and identify the threshold BP for initiating treatment. The exact incidence of hypertension in very low birth weight (VLBW) infants at corrected term gestation is difficult to estimate due to lack of a robust and universally accepted definition for hypertension. Current blood pressure (BP) nomograms for VLBW infants are based on a limited number of VLBW infants followed to term corrected gestational age (CGA). There is a need for a large multicenter study to develop more appropriate gestational age or birth weight based BP centiles with chronological data from birth through term corrected gestation age, infancy and beyond.There is emerging evidence that preterm birth is associated with increased risk for hypertension later in life -4. A lare BP nomogram, for infants admitted to NICU, was derived from a cohort, mostly consisting of late preterm or term infants   during their prolonged neonatal intensive care unit (NICU) stay and are at risk for developing chronic lung disease , which can potentially elevate their BP . The mosoup1995) . From ououp1995) , we specThe objective of this study was to determine the clinical utility of the Zubrow curves in diagnosis of hypertension in VLBW infants at their term CGA.MetroHealth NICU is a level III unit with 50 beds serving a diverse population from Cleveland and North East Ohio with over 600 admissions per year including approximately 100-150 VLBW infants/year. Majority of NICU admissions are inborn. This is a retrospective case control study of all VLBW infants admitted to the NICU between March 2009 and May 2011. Cases were defined as VLBW infants who were required to stay in the NICU at CGA of 40 weeks. Controls were defined as term infants of 37-40 weeks of gestation who required admission to the NICU and remained in the NICU at the CGA of 40 weeks. For comparison, the controls were selected in a manner similar to the term infants included in the Zubrow’s study cohort  .BP data The study protocol was in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of MetroHealth Medical Center, Cleveland, Ohio, United States.P value <0.05 was considered statistically significant. The statistical software IBM SPSS Statistics version 19  was used for the statistical analysis of the data.Two-way analysis of variance (ANOVA)  was perfFrom consecutive 200 eligible VLBW and term infant cohorts admitted during the study period, 75 VLBW infants (cases) and 69 term infants (controls) met the inclusion criteria. Remainder of the infants was either excluded, discharged or had expired before reaching the CGA of 40 weeks.P=0.002), African American , have lower body weight at 40 weeks CGA , higher exposure to central line days , urinary tract infection  and culture positive bacteremia . There was no significant difference in exposure to maternal preeclampsia, postnatal steroids and acute kidney injury between VLBW infants and term infants. 27/75 (36.0%) of the VLBW infants and 12/69 (17.4%) of the term infants were categorized as unstable (P=0.15). The incidence of CLD in VLBW infants admitted during the study period was 33% (66/200), but the incidence was higher in the selected case group of VLBW infants who were required to stay in the NICU at 40 weeks CGA (63% [47/75]).VLBW infants , compared to term infants , were more likely to be small for gestational age  and mean MBP , but similar DBP  , MAP (72.40 versus 67.5), and DBP (59.2 vs. 56.5)  . The ress. 56.5) . The 95ts. 56.5)  (8). In P≤0.001), lower gestational age at birth , but had similar weight  and BP  at 40 weeks CGA.In VLBW infants who were diagnosed with hypertension, the average SBP at CGA of 40 weeks was 100 mm Hg. Amongst the 75 VLBW infants, infants with CLD (47/75) compared to infants without CLD (28/75) had lower birth weight  were developed in 1994 based on a prospective, multicenter study of 608 neonates admitted to 14 different NICUs in Philadelphia over the first 99 days of life ,8,10. InDionne et al have constructed a clinically useful reference table of estimated BP values  after the first 2 weeks of age in infants from 26 to 44 weeks CGA based on previous published studies ,10,15-19The organs that develop late in-utero, such as kidneys, lungs or eyes, are most at risk of altered remodeling and development in the prematurely born sick newborn. Nephrogenesis is completed by 34-35 weeks gestation . Final nThe VLBW infants in our cohort were sick enough to continue in the NICU at 40 weeks CGA and a significant number had CLD. CLD in premature infants has been previously reported to be associated with higher incidence of hypertension and more than half of these were diagnosed with hypertension after NICU discharge ,12,25. CIn spite of having lower body weights, VLBW infants at 40 weeks corrected gestation, have higher systolic and mean BP compared to term infants, which exposes them to an elevated risk for cardiovascular morbidities later in life. Preterm infants especially VLBW infants are at higher risk of developing hypertension as a result of early life postnatal physiological programming, deficits in nephron endowment and exposure to adverse environmental events during their NICU stay. Currently available BP nomograms for VLBW infants are based on a limited number of VLBW infants followed to term CGA. The exact incidence of hypertension in VLBW infants at corrected term gestation is difficult to estimate due to lack of a robust and universally accepted definition for hypertension. There is a need for a large multicenter study to develop more appropriate gestational age or birth weight based BP centiles with chronological data from birth through term corrected gestation age, infancy and beyond. VLBW infants should be monitored for elevated BP and altered renal function throughout infancy, childhood and adulthood.Our study has several limitations. It is retrospective case control study from a single center NICU. Although, we have a written NICU protocol for BP monitoring, BP documented in the chart may be have been affected by infant temperament, feeding and infant position. Although our protocol requires it and frequently the nursing documentation comments upon these factors, it is conceivable that documentation may not have been 100% compliant. BP measurement using oscillometric manometers may be less accurate than the gold standard intra-arterial monitors but studies have shown good correlation between oscillometric and umbilical artery BP in neonates . Though This study was made possible by the Edward M. Chester MD Summer Scholars Program at Metro Health Medical Center.All the authors have contributed towards performing the study and preparation of the manuscript and they all have approved the latest version of the article. SV and DK conceptualized the study, participated in data collection, analysis-interpretation, manuscript writing and editing. CS, SO, and NP assisted in data collection, analysis, manuscript writing and editing. DMS helped finalize the methodology, data analysis and interpretation and manuscript editing. JD helped with compiling document elements for submission and manuscript editing. RR helped with data analysis and interpretation, manuscript preparation and editing.The authors have no financial relationships relevant to this article to disclose.Ethical issues  have been completely observed by the authors.The authors have no conflicts of interest to disclose.