Characterisation of Staphylococci species from neonatal blood cultures in low- and middle-income countries.

BACKGROUND: In low- and middle-income countries (LMIC) Staphylococcus aureus is regarded as one of the leading bacterial causes of neonatal sepsis, however there is limited knowledge on the species diversity and antimicrobial resistance caused by Gram-positive bacteria (GPB). METHODS: We characterised GPB isolates from neonatal blood cultures from LMICs in Africa (Ethiopia, Nigeria, Rwanda, and South Africa) and South-Asia (Bangladesh and Pakistan) between 2015-2017. We determined minimum inhibitory concentrations and performed whole genome sequencing (WGS) on Staphylococci isolates recovered and clinical data collected related to the onset of sepsis and the outcome of the neonate up to 60 days of age. RESULTS: From the isolates recovered from blood cultures, Staphylococci species were most frequently identified. Out of 100 S. aureus isolates sequenced, 18 different sequence types (ST) were found which unveiled two small epidemiological clusters caused by methicillin resistant S. aureus (MRSA) in Pakistan (ST8) and South Africa (ST5), both with high mortality (n = 6/17). One-third of S. aureus was MRSA, with methicillin resistance also detected in Staphylococcus epidermidis, Staphylococcus haemolyticus and Mammaliicoccus sciuri. Through additional WGS analysis we report a cluster of M. sciuri in Pakistan identified between July-November 2017. CONCLUSIONS: In total we identified 14 different GPB bacterial species, however Staphylococci was dominant. These findings highlight the need of a prospective genomic epidemiology study to comprehensively assess the true burden of GPB neonatal sepsis focusing specifically on mechanisms of resistance and virulence across species and in relation to neonatal outcome.

Impact of interpregnancy intervals on perinatal and neonatal outcomes in a multiethnic Pakistani population.

BACKGROUND: Short birth intervals (SBIs) and long birth intervals (LBIs) have been shown to have serious implications for health of both mothers and their children. This study was aimed to investigate the determinants and reproductive outcome of SBI and LBI in a multiethnic Pakistani population. METHODS: In a cross-sectional prospective study design, 2798 women admitted in a tertiary-care hospital in Islamabad for delivery were recruited and data on second or higher birth order deliveries were collected. Birth intervals were defined as short (<24 months) and long (>36 months). The reproductive outcome was defined in terms of perinatal and neonatal mortalities, and neonatal complications. Univariate and multivariate logistic regression analyses were performed. RESULTS: Pregnancies with SBI and LBI were observed in 20% and 24% of 2798 women, respectively. Women with SBI had increased odds of perinatal death [adjusted odd ratio (AOR): 1.50] and neonatal death (AOR: 1.47) as compared to women with optimal birth intervals, while women with LBI had slightly lower odds of perinatal deaths (AOR: 0.96), but increased odds of neonatal deaths (AOR: 1.12). Further, the pregnancies with both SBI and LBI were associated with increased odds of short body length, low birth weight, small head circumference and low APGAR score. CONCLUSION: Nearly half of all pregnancies do not have optimal birth spacing albeit there is wide heterogeneity in the distribution of BI in various Pakistani ethnicities. Pregnancies with SBI and LBI had high risk of adverse reproductive outcome. Intervention programs for maternal and child health need to emphasize optimal birth spacing.

Birth interval (BI) or interpregnancy interval is the length of time between a birth and conception of the next pregnancy. Short birth intervals (SBIs) as well as long birth intervals (LBIs) have been shown to have serious implications for health of both mothers and their children. WHO recommendation for optimal spacing between 3 and 5 years. In this study, we aimed to investigate the effect of SBI and LBI on pregnancy outcome in the Pakistani population. A total of 2798 pregnant women admitted in a tertiary-care hospital in Islamabad for delivery were recruited and data on BI and pregnancy outcomes, i.e. perinatal and neonatal mortalities, and neonatal complications, were obtained. Results revealed that pregnancies with SBI and LBI were 20% and 24% of the total pregnancies, respectively. Women with SBI had higher likelihood of perinatal and neonatal death as compared to women with optimal birth intervals. Similarly, the women with LBI had higher likelihood of neonatal deaths. Furthermore, the pregnancies with both SBI and LBI were associated neonatal complications like short body length, low birth weight, small head circumference and low APGAR score. In conclusion, nearly half of all pregnancies do not have optimal birth spacing. Intervention programs for maternal and child health need to emphasize optimal birth spacing.

Colonisation of hospital surfaces from low- and middle-income countries by extended spectrum ß-lactamase- and carbapenemase-producing bacteria.

Hospital surfaces can harbour bacterial pathogens, which may disseminate and cause nosocomial infections, contributing towards mortality in low- and middle-income countries (LMICs). During the BARNARDS study, hospital surfaces from neonatal wards were sampled to assess the degree of environmental surface and patient care equipment colonisation by Gram-negative bacteria (GNB) carrying antibiotic resistance genes (ARGs). Here, we perform PCR screening for extended-spectrum ß-lactamases (blaCTX-M-15) and carbapenemases (blaNDM, blaOXA-48-like and blaKPC), MALDI-TOF MS identification of GNB carrying ARGs, and further analysis by whole genome sequencing of bacterial isolates. We determine presence of consistently dominant clones and their relatedness to strains causing neonatal sepsis. Higher prevalence of carbapenemases is observed in Pakistan, Bangladesh, and Ethiopia, compared to other countries, and are mostly found in surfaces near the sink drain. Klebsiella pneumoniae, Enterobacter hormaechei, Acinetobacter baumannii, Serratia marcescens and Leclercia adecarboxylata are dominant; ST15 K. pneumoniae is identified from the same ward on multiple occasions suggesting clonal persistence within the same environment, and is found to be identical to isolates causing neonatal sepsis in Pakistan over similar time periods. Our data suggests persistence of dominant clones across multiple time points, highlighting the need for assessment of Infection Prevention and Control guidelines.

Neonatal sepsis and mortality in low-income and middle-income countries from a facility-based birth cohort: an international multisite prospective observational study.

BACKGROUND: Neonatal sepsis is a primary cause of neonatal mortality and is an urgent global health concern, especially within low-income and middle-income countries (LMICs), where 99% of global neonatal mortality occurs. The aims of this study were to determine the incidence and associations with neonatal sepsis and all-cause mortality in facility-born neonates in LMICs. METHODS: The Burden of Antibiotic Resistance in Neonates from Developing Societies (BARNARDS) study recruited mothers and their neonates into a prospective observational cohort study across 12 clinical sites from Bangladesh, Ethiopia, India, Pakistan, Nigeria, Rwanda, and South Africa. Data for sepsis-associated factors in the four domains of health care, maternal, birth and neonatal, and living environment were collected for all mothers and neonates enrolled. Primary outcomes were clinically suspected sepsis, laboratory-confirmed sepsis, and all-cause mortality in neonates during the first 60 days of life. Incidence proportion of livebirths for clinically suspected sepsis and laboratory-confirmed sepsis and incidence rate per 1000 neonate-days for all-cause mortality were calculated. Modified Poisson regression was used to investigate factors associated with neonatal sepsis and parametric survival models for factors associated with all-cause mortality. FINDINGS: Between Nov 12, 2015 and Feb 1, 2018, 29 483 mothers and 30 557 neonates were enrolled. The incidence of clinically suspected sepsis was 166·0 (95% CI 97·69-234·24) per 1000 livebirths, laboratory-confirmed sepsis was 46·9 (19·04-74·79) per 1000 livebirths, and all-cause mortality was 0·83 (0·37-2·00) per 1000 neonate-days. Maternal hypertension, previous maternal hospitalisation within 12 months, average or higher monthly household income, ward size (>11 beds), ward type (neonatal), living in a rural environment, preterm birth, perinatal asphyxia, and multiple births were associated with an increased risk of clinically suspected sepsis, laboratory-confirmed sepsis, and all-cause mortality. The majority (881 [72·5%] of 1215) of laboratory-confirmed sepsis cases occurred within the first 3 days of life. INTERPRETATION: Findings from this study highlight the substantial proportion of neonates who develop neonatal sepsis, and the high mortality rates among neonates with sepsis in LMICs. More efficient and effective identification of neonatal sepsis is needed to target interventions to reduce its incidence and subsequent mortality in LMICs. FUNDING: Bill & Melinda Gates Foundation.

Effects of antibiotic resistance, drug target attainment, bacterial pathogenicity and virulence, and antibiotic access and affordability on outcomes in neonatal sepsis: an international microbiology and drug evaluation prospective substudy (BARNARDS).

BACKGROUND: Sepsis is a major contributor to neonatal mortality, particularly in low-income and middle-income countries (LMICs). WHO advocates ampicillin-gentamicin as first-line therapy for the management of neonatal sepsis. In the BARNARDS observational cohort study of neonatal sepsis and antimicrobial resistance in LMICs, common sepsis pathogens were characterised via whole genome sequencing (WGS) and antimicrobial resistance profiles. In this substudy of BARNARDS, we aimed to assess the use and efficacy of empirical antibiotic therapies commonly used in LMICs for neonatal sepsis. METHODS: In BARNARDS, consenting mother-neonates aged 0-60 days dyads were enrolled on delivery or neonatal presentation with suspected sepsis at 12 BARNARDS clinical sites in Bangladesh, Ethiopia, India, Pakistan, Nigeria, Rwanda, and South Africa. Stillborn babies were excluded from the study. Blood samples were collected from neonates presenting with clinical signs of sepsis, and WGS and minimum inhibitory concentrations for antibiotic treatment were determined for bacterial isolates from culture-confirmed sepsis. Neonatal outcome data were collected following enrolment until 60 days of life. Antibiotic usage and neonatal outcome data were assessed. Survival analyses were adjusted to take into account potential clinical confounding variables related to the birth and pathogen. Additionally, resistance profiles, pharmacokinetic-pharmacodynamic probability of target attainment, and frequency of resistance (ie, resistance defined by in-vitro growth of isolates when challenged by antibiotics) were assessed. Questionnaires on health structures and antibiotic costs evaluated accessibility and affordability. FINDINGS: Between Nov 12, 2015, and Feb 1, 2018, 36 285 neonates were enrolled into the main BARNARDS study, of whom 9874 had clinically diagnosed sepsis and 5749 had available antibiotic data. The four most commonly prescribed antibiotic combinations given to 4451 neonates (77·42%) of 5749 were ampicillin-gentamicin, ceftazidime-amikacin, piperacillin-tazobactam-amikacin, and amoxicillin clavulanate-amikacin. This dataset assessed 476 prescriptions for 442 neonates treated with one of these antibiotic combinations with WGS data (all BARNARDS countries were represented in this subset except India). Multiple pathogens were isolated, totalling 457 isolates. Reported mortality was lower for neonates treated with ceftazidime-amikacin than for neonates treated with ampicillin-gentamicin (hazard ratio [adjusted for clinical variables considered potential confounders to outcomes] 0·32, 95% CI 0·14-0·72; p=0·0060). Of 390 Gram-negative isolates, 379 (97·2%) were resistant to ampicillin and 274 (70·3%) were resistant to gentamicin. Susceptibility of Gram-negative isolates to at least one antibiotic in a treatment combination was noted in 111 (28·5%) to ampicillin-gentamicin; 286 (73·3%) to amoxicillin clavulanate-amikacin; 301 (77·2%) to ceftazidime-amikacin; and 312 (80·0%) to piperacillin-tazobactam-amikacin. A probability of target attainment of 80% or more was noted in 26 neonates (33·7% [SD 0·59]) of 78 with ampicillin-gentamicin; 15 (68·0% [3·84]) of 27 with amoxicillin clavulanate-amikacin; 93 (92·7% [0·24]) of 109 with ceftazidime-amikacin; and 70 (85·3% [0·47]) of 76 with piperacillin-tazobactam-amikacin. However, antibiotic and country effects could not be distinguished. Frequency of resistance was recorded most frequently with fosfomycin (in 78 isolates [68·4%] of 114), followed by colistin (55 isolates [57·3%] of 96), and gentamicin (62 isolates [53·0%] of 117). Sites in six of the seven countries (excluding South Africa) stated that the cost of antibiotics would influence treatment of neonatal sepsis. INTERPRETATION: Our data raise questions about the empirical use of combined ampicillin-gentamicin for neonatal sepsis in LMICs because of its high resistance and high rates of frequency of resistance and low probability of target attainment. Accessibility and affordability need to be considered when advocating antibiotic treatments with variance in economic health structures across LMICs. FUNDING: The Bill & Melinda Gates Foundation.

Characterization of antimicrobial-resistant Gram-negative bacteria that cause neonatal sepsis in seven low- and middle-income countries.

Antimicrobial resistance in neonatal sepsis is rising, yet mechanisms of resistance that often spread between species via mobile genetic elements, ultimately limiting treatments in low- and middle-income countries (LMICs), are poorly characterized. The Burden of Antibiotic Resistance in Neonates from Developing Societies (BARNARDS) network was initiated to characterize the cause and burden of antimicrobial resistance in neonatal sepsis for seven LMICs in Africa and South Asia. A total of 36,285 neonates were enrolled in the BARNARDS study between November 2015 and December 2017, of whom 2,483 were diagnosed with culture-confirmed sepsis. Klebsiella pneumoniae (n = 258) was the main cause of neonatal sepsis, with Serratia marcescens (n = 151), Klebsiella michiganensis (n = 117), Escherichia coli (n = 75) and Enterobacter cloacae complex (n = 57) also detected. We present whole-genome sequencing, antimicrobial susceptibility and clinical data for 916 out of 1,038 neonatal sepsis isolates (97 isolates were not recovered from initial isolation at local sites). Enterobacterales (K. pneumoniae, E. coli and E. cloacae) harboured multiple cephalosporin and carbapenem resistance genes. All isolated pathogens were resistant to multiple antibiotic classes, including those used to treat neonatal sepsis. Intraspecies diversity of K. pneumoniae and E. coli indicated that multiple antibiotic-resistant lineages cause neonatal sepsis. Our results will underpin research towards better treatments for neonatal sepsis in LMICs.

Genome-wide analysis of p53 under hypoxic conditions.

Hypoxia is an important nongenotoxic stress that modulates the tumor suppressor activity of p53 during malignant progression. In this study, we investigated how genotoxic and nongenotoxic stresses regulate p53 association with chromatin, p53 transcriptional activity, and p53-dependent apoptosis. We found that genotoxic and nongenotoxic stresses result in the accumulation and binding of the p53 tumor suppressor protein to the same cognate binding sites in chromatin. However, it is the stress that determines whether downstream signaling is mediated by association with transcriptional coactivators. In contrast to p53 induced by DNA-damaging agents, hypoxia-induced p53 has primarily transrepression activity. Using extensive microarray analysis, we identified families of repressed targets of p53 that are involved in cell signaling, DNA repair, cell cycle control, and differentiation. Following our previous study on the contribution of residues 25 and 26 to p53-dependent hypoxia-induced apoptosis, we found that residues 25-26 and 53-54 and the polyproline- and DNA-binding regions are also required for both gene repression and the induction of apoptosis by p53 during hypoxia. This study defines a new role for residues 53 and 54 of p53 in regulating transrepression and demonstrates that 25-26 and 53-54 work in the same pathway to induce apoptosis through gene repression.

Less Invasive Surfactant Administration in Preterm Infants with Respiratory Distress Syndrome.

OBJECTIVE: To compare the need of mechanical ventilation between LISA (less invasive surfactant administration) method and conventional INSURE method (INtubation SURfactant administration and Extubation) in spontaneously breathing preterm infants with respiratory distress syndrome (RDS). STUDY DESIGN: An experimental study. PLACE AND DURATION OF STUDY: Department of Neonatology, PIMS, Islamabad, from April to December 2017. METHODOLOGY: A total of 100 preterm infants <34 weeks gestation, on nasal CPAP requiring fraction of inspire oxygen (FiO2) >0.4, with respiratory distress syndrome (RDS) were included in the study and divided randomly into two groups, 50 each. RESULTS: There were 28 (56%) males in LISA and 31 (62%) in the INSURE group. Median birth weight was 1300 grams (IQR 600) in LISA, while 1400 grams (IQR 400) in INSURE infants. C-section rate was 52% (n=26) and 48% (n=24) in LISA and INSURE, respectively. Pre-natal steroids were given to 38 patients (76%) in LISA and 30 patients (60%) in INSURE group. LISA patients had significantly less need of mechanical ventilation with p-value <0.05 {30% (n=15) vs. 60% (n=30)}. The median duration of mechanical ventilation was 40 hours (IQR 75) and 71 hours (IQR 62) in LISA and INSURE, respectively. Similarly, median FiO2 reduction was 30 (IQR 30) in LISA group and it was 25 (IQR 10) in INSURE group, with p-value <0.05. There was no significant difference in mortality, hospital stay and complications. CONCLUSION: LISA technique was safe, non-invasive approach of surfactant administration, with reduced need of mechanical ventilation rate and duration.

Quantification of motion of different thoracic locations using four-dimensional computed tomography: implications for radiotherapy planning.

PURPOSE: To assess the respiratory motion of different thoracic nodal locations and its dependence on the presence of enlarged nodes; to assess the respiratory motion of different parenchymal tumor locations; and to determine the appropriate margins to cover the respiratory motion of targets at these locations. METHODS AND MATERIALS: We reviewed the four-dimensional computed tomography scans of 20 patients with thoracic tumors treated at our institution. The motion of four central thoracic locations (aortic arch, carina, and bilateral hila), parenchymal tumor locations (upper vs. lower, and anterior vs. middle vs. posterior thorax), and bilateral diaphragmatic domes was measured. RESULTS: For the central thoracic locations, the largest motion was in the superoinferior (SI) dimension (>5 mm for bilateral hila and carina, but <4 mm for aortic arch). No significant difference was found in the motion of these locations in the absence or presence of enlarged nodes. For parenchymal tumors, upper tumors exhibited smaller SI motion than did lower tumors (3.7 vs. 10.4 mm, p = 0.029). Similarly, anterior tumors exhibited smaller motion than did posterior tumors in both the SI (4.0 vs. 8.0 mm, p = 0.013) and lateral (2.8 vs. 4.6 mm, p = 0.045) directions. The margins that would be needed to encompass the respiratory motion of each of the evaluated locations in 95% of patients were tabulated and range from 3.4 to 37.2 mm, depending on the location and direction. CONCLUSIONS: The results of our study have provided data for appropriate site-specific internal target volume expansion that could be useful in the absence of four-dimensional computed tomography-based treatment planning. However, generalizing the results from a small patient population requires discretion.

Stereotactic radiosurgery and fractionated stereotactic radiotherapy in the treatment of uveal melanoma.

Uveal melanoma is the most common primary intraocular malignant tumor. Radiation therapy has now replaced enucleation as the treatment of choice, with radioactive eye plaques and proton therapy being the two most studied radiotherapy modalities. More recently, stereotactic radiosurgery and fractionated stereotactic radiotherapy have emerged as promising, non-invasive treatments for uveal melanoma. This review summarizes the available literature on these newer treatment modalities.

A comparison study of different PCR assays in measuring circulating plasma epstein-barr virus DNA levels in patients with nasopharyngeal carcinoma.

PURPOSE: To compare the performance of three PCR assays in measuring circulating Epstein-Barr virus (EBV). DNA levels in nasopharyngeal carcinoma patients and to confirm its prognostic significance. EXPERIMENTAL DESIGN: Plasma from 58 newly diagnosed nasopharyngeal carcinoma patients were collected before, during, and every 3 to 6 months after radiotherapy. EBV DNA levels were determined by real-time quantitative PCR using primer/probe sets for polymerase-1 (Pol-1), latent membrane protein 2 (Lmp2), and BamHI-W. Pretreatment levels from the three assays were correlated with each other and serial measurements from the Pol-1 assay were correlated with clinical variables. RESULTS: Pol-1 was more accurate than BamHI-W in predicting EBV DNA concentrations in cell lines. Of the three assays, BamHI-W yielded the highest concentrations followed by Pol-1 in plasmas (n = 23). The correlation coefficient was 0.99 (P < 0.0001) for Pol-1 and Lmp2, 0.66 (P < 0.0001) for Pol-1 and BamHI-W, and 0.55 (P < 0.0001) for BamHI-W and Lmp2. Elevated pretreatment DNA levels as detected by Pol-1 were correlated with advanced nodal stage (P = 0.04) and overall stage (P = 0.028). There was no correlation between pretreatment EBV DNA levels and freedom-from-relapse or overall survival; however, there was a significant correlation between posttreatment levels and these variables. The 2-year freedom-from-relapse and overall survival rates were 92% and 94% for patients with undetectable, and 37% and 55% for those with detectable, posttreatment levels (P < 0.0001 and P < 0.002). CONCLUSIONS: The three PCR assays yielded similar results in detecting EBV DNA in plasmas. The Pol-1-detected posttreatment EBV DNA level was the strongest predictor for treatment outcomes.

Residents' educational needs during transition into radiation oncology residency.

PURPOSE: The aim of this study was to assess current practices, strengths, and deficiencies in the orientation process for incoming radiation oncology (RO) residents. METHODS: An institutional review board-approved anonymous survey was distributed electronically to RO residents in postgraduate years 2 to 5 and those in their first postgraduate years. Questions were included on the type and utility of orientation materials received by residents before and upon entering RO residency. RESULTS: Responses were received from 25.3% of all current and recent residents. Most residents (81.3%) had 2 or 3 months of prior experience rotating in clinical RO. Orientation materials in RO were received by 74.1% of residents before starting residency. An orientation at the start of RO residency was received by 95.4% of RO residents. Orientation length was <1 hour in 2.8%, 1 hour to a half day in 7.8%, more than a half day but <1 full day in 21.8%, >1 full day but <1 week in 45.8%, and >1 week but <1 month in 20.1%. Almost half of RO residents (48.4%) felt that an RO orientation was essential, but only 11.3% of residents felt that their orientation programs were essential. A statistically significant Spearman's correlation was observed between programs with longer orientation and increased helpfulness of orientation (ρ = 0.26, P = .008). Residents with more components in their onsite orientations felt that their orientations were more helpful (ρ = 0.407, P < .001). CONCLUSIONS: Radiation oncology residents could benefit from a more comprehensive orientation, including a broader array of materials sent to incoming residents for their review before starting residency and a more extensive onsite orientation.

Combined modality approaches in the management of adult glioblastoma.

Over the past two decades, management of newly diagnosed glioblastoma has undergone significant evolution. While surgery has long been a mainstay of management for this disease, and while radiotherapy has a proven survival role, initial efforts at radiotherapy dose escalation, use of radiosurgery, brachytherapy, and altered fractionation did not improve patient survival. Recently, multiple modality therapy integrating maximal safe resection, postoperative radiation, and new systemic therapies have resulted in improved patient outcomes compared with older regimens utilizing surgery and postoperative radiation alone. Numerous trials are currently underway investigating the combination of surgery, radiation, and systemic therapy with targeted agents to find ways to further improve outcomes for adults with glioblastoma.

Advanced-staged tonsillar squamous carcinoma: organ preservation versus surgical management of the primary site.

BACKGROUND: Our aim was to review our experience in the management of advanced tonsillar squamous cell carcinoma (SCC) and to compare treatment outcomes between patients treated with and without surgery to the primary site. METHODS: The records of 74 patients with advanced-stage tonsillar SCC were reviewed. The median age at diagnosis was 58 years. Thirty-eight patients received definitive surgery to the primary site, and 36 were treated with an organ-preservation approach (OP) using radiotherapy +/- chemotherapy. RESULTS: No significant difference in overall survival (OS) or freedom from relapse (FFR) by treatment was found. T classification and N status were significant independent predictors on multivariate analysis for OS and FFR. Major late toxicity was noted in 10 patients in the surgical group and nine in the OP group. CONCLUSION: Patients treated with OP and primary surgery had comparable OS and FFR. T classification and N status were significant independent predictors for tumor relapse and survival. On the basis of these results, we favor organ-preservation therapy for patients with advanced-stage tonsillar SCC.

Regulation of ribosomal S6 kinase 2 by mammalian target of rapamycin.

Phosphorylation of the ribosomal S6 subunit is tightly correlated with enhanced translation initiation of a subset of mRNAs that encodes components of the protein synthesis machinery, which is an important early event that controls mammalian cell growth and proliferation. The recently identified S6 kinase 2 (S6K2), together with its homologue S6K1, is likely responsible for the mitogen-stimulated phosphorylation of S6. Like S6K1, the activation of S6K2 requires signaling from both the phosphatidylinositol 3-kinase and the mammalian target of rapamycin (mTOR). Here we report the investigation of the mechanisms of S6K2 regulation by mTOR. We demonstrate that similar to S6K1 the serum activation of S6K2 in cells is dependent on mTOR kinase activity, amino acid sufficiency, and phosphatidic acid. Previously we have shown that mTOR is a cytoplasmic-nuclear shuttling protein. As a predominantly nuclear protein, S6K2 activation was facilitated by enhanced mTOR nuclear import with the tagging of an exogenous nuclear localization signal and diminished by enhanced mTOR nuclear export with the tagging of a nuclear export sequence. However, further increase of mTOR nuclear import by the tagging of four copies of nuclear localization signal resulted in its decreased ability to activate S6K2, suggesting that mTOR nuclear export may also be an integral part of the activation process. Consistently, the nuclear export inhibitor leptomycin B inhibited S6K2 activation. Taken together, our observations suggest a novel regulatory mechanism in which an optimal cytoplasmic-nuclear distribution or shuttling rate for mTOR is required for maximal activation of the nuclear S6K2.