Breast Cancer (BC) is a highly prevalent disease. A woman living in the United States has a 12.3% lifetime risk of being diagnosed with breast cancer [HER2 is a member of the epidermal growth factor receptor (EGFR) family. Other family members include EGFR or HER1, HER3 and HER4. HER2 can form heterodimers with any of the other three receptors, and is considered to be the preferred dimerization partner of the other HER or ErbB receptors .In addition to being an important prognostic factor in women diagnosed with BC, HER2 overexpression also identifies those patients who benefit from treatment with agents that target HER2, such as trastuzumab, pertuzumab, trastuzumab emtansine (T-DM1) and small molecules tyrosine kinase inhibitors of HER2 , 11, 127This review paper will concentrate on major biological pathways that ultimately lead to resistance to anti-HER2 therapies in BC, summarizing their mechanisms. Strategies to overcome this resistance, and the rationale involved in each tactics to revert this scenario will be presented to the reader. Since the approval of trastuzumab in the adjuvant setting by the FDA in 2006, most patients with HER2+ early BC are doing very well with standard trastuzumab therapy added to chemotherapy. Planned Joint Analysis of OS from NSABP B-31 and NCCTG N9831, demonstrated that the addition of trastuzumab to a taxane and anthracycline chemotherapy backbone resulted in a durable improvement in survival, after a median time on study of approximately eight and a half years. 2 and trastuzumab at 2 mg/kg for 12 weeks, followed by 9 months of trastuzumab in 406 women with HER2-positive, node-negative tumors ≤ 3 cm. Disease-free survival achieved an impressive result at 3 years of 98.7% (P < .0001), and the regimen was associated with great cardiac safety. [In fact, patients nearly diagnosed with localized HER2+ BC have an excellent prognosis, even if treated with less toxic adjuvant systemic therapy, as recently demonstrated by Dana Farber investigators . This noIn the metastatic setting, Slamon et al.  evaluateIncorporation of new agents, as evidenced by the CLEOPATRA trial, in which pertuzumab, a humanized monoclonal antibody that binds to HER2 at a different epitope than that at which trastuzumab binds, was added to the standard docetaxel and trastuzumab combination, and lead to striking improvements in PFS and OS in a cohort of advanced HER2+ BC patients, reaching the median OS boundary of almost 5 years. –15Despite this robust clinical benefit, anti-HER therapy resistance, either de novo or acquired, is an important clinical challenge in the management of BC patients. Research has been dedicated to a better understanding of the molecular mechanisms involved of trastuzumab resistance. The PI3K/AKT/mTOR pathway is an important growth factor pathway and a key effector of HER2 signalling. HER2 phosphorylation may lead to pathway activation. ConstituMany investigators evaluated trastuzumab benefit in patients enrolled in clinical trials in distinct disease scenarios, according to alterations in the PI3K pathway. Most of them failed to demonstrate a relationship between PIK3CA mutations and trastuzumab benefit. As an example, the FinHER adjuvant phase III trial genotyped 687 HER2+ BC patients. PIK3CA mutations were not statistically significantly associated with trastuzumab benefit, or survival outcomes.  SimilarlThe EMILIA trial compared the effectiveness of TDM-1 versus lapatinib and capecitabine in patients previously treated with trastuzumab. Samples from patients were prospectively collected for PIK3CA mutation analysis. Patients in the lapatinib arm with PIK3CA mutations had worse outcomes than those with wild-type PIK3CA, but the presence of PIK3CA mutations had absolutely no effect on PFS or OS in patients treated with T-DM1, suggesting that this drug might be an attractive alternative for patients harbouring this alteration. in vitro studies that initially identified PIK3CA mutation as a resistance factor for HER2-targeted treatment did not account for trastuzumab mediated antibody dependent cellular cytotoxicity (ADCC). Recognition of tumor cells opsonized by monoclonal antibodies, such as trastuzumab, is mediated through receptors expressed on effector cells, as well as monocytes, dendritic cells, and granulocytes. Upon recognition, these effectors induce tumor cell death. Dendritic cells capture the monoclonal antibody conjugated with tumor antigens released by dying cells in the form of immune complexes. Ultimately, these processed cells are presented to cytotoxic and helper T cells. As a consequence, both tumor-specific cytotoxic T cells and T-helper cells are activated, leading to tumor B-cell stimulation and expansion. The production of tumor-directed host antibodies is the final step in this important ADCC cytotoxic mechanism. [in vitro or in immunosuppressed models. Furthermore, trastuzumab is a human monoclonal antibody and therefore, unlikely to engender an immune reaction in a model system in vitro.The evidence described above is somehow contradictory to preclinical data. One important aspect to be taken into account is the fact that chanism.  Also, trchanism.  In fact,chanism.  PrecliniSurvival outcomes of HER2+ BC patients might be influenced by PIK3CA status. Biomarker analysis from the CLEOPATRA trial demonstrated that PIK3CA mutation was associated with worse survival outcomes among patients with advanced HER2+ BC. NevertheAdditionally, a group of researchers from Memorial Sloan-Kettering Cancer Center evaluated a group of 63 patients with HER2+ BC with disease recurrence after adjuvant trastuzumab treatment, or progressive metastatic disease on a trastuzumab-containing regimen. All patiThese authors also compared PTEN loss and PIK3CA mutation rate between 2 HER2+ BC cohorts of patients: one of them refractory to trastuzumab and the other unexposed to trastuzumab. They demonstrated that the combined rate of PTEN loss and PIK3CA mutation in the trastuzumab-refractory tumors was 71%, compared with 44% in the unexposed cohort, suggesting that trastuzumab exposure may lead to alterations in PIK3CA pathway.Therefore, evidence supporting the hypothesis that PI3K alterations might lead to worse prognosis in HER2+ BC, as well as resistance to anti-HER2 therapy, is mostly based on small retrospective series. At the present time, the first (and still only) actionable genomic alteration in BC, is amplification of HER2, and anti-HER2 therapy is required in all disease scenarios where this alteration is observed.Correlation between PI3K alterations and pCR achievement was extensively studied. Loibl and colleagues investigated whether the presence of a PIK3CA mutation affected the odds of achieving a pCR among patients enrolled in the GeparQuattro, GeparQuinto, and GeparSixto neoadjuvant clinical trials.  In thesep < 0.001), for patients with and without a PIK3CA mutation, respectively. [Of note, at the 2015 American Society of Clinical Oncology (ASCO) Meeting, investigators reported pCR rates according to PIK3CA status among HER2+ BC patients who received neoadjuvant trastuzumab, lapatinib, or both in addition to a taxane-based chemotherapy. Significantly lower rates of pCR were observed in PIK3CA mutant tumors after anti-HER2 treatment, namely 16.2% versus 29.6% (ctively. Table p=0.0067).[Therefore, much interest is focused on this important proliferative pathway that may contribute to trastuzumab resistance. Inhibition of mTOR has been shown to be an important therapeutic strategy in HER2+ BC. BOLERO-3 aimed to assess whether the addition of the mTOR inhibitor everolimus to trastuzumab might restore sensitivity to trastuzumab. Eligible patients for this trial had HER2+, trastuzumab-resistant, advanced BC who had previously received taxane therapy. Patients were randomized to receive daily everolimus (5 mg/day) plus weekly trastuzumab (2 mg/kg) and vinorelbine (25 mg/m2) or to placebo plus trastuzumab plus vinorelbine, in 3-week cycle. As a result, the addition of everolimus prolonged PFS from 5.78 months to 7.00 months . In PFS sBOLERO-1 also focuses on the strategy of targeting the PI3K/AKT/mTOR pathway. In this trial, everolimus was added to paclitaxel and trastuzumab in patients with metastatic HER2+ BC patients who had not received previous trastuzumab or chemotherapy for advanced BC within 12 months of randomisation. Primary endpoint was PFS, and secondary endpoints included OS, response rate (RR), and clinical benefit rate. The results were presented at the 2014 San Antonio Breast Cancer Symposium (SABCS) and published in Lancet Oncology. The studConsidering that mTOR inhibitors add a substantial amount of toxicity to therapy, biomarkers to identify patients who could derive more benefit from therapy are being studied. At the 2015 ASCO Meeting, researchers evaluated tumor tissue from patients recruited in BOLERO-1 and BOLERO-3. Exons of 282 cancer related genes were analyzed by next generation sequencing (NGS), and PTEN levels were evaluated by immunohistochemistry. Hyperactive PI3K pathway was defined as low PTEN levels or mutations in PIK3CA pathway. In both trials, patients with hyperactive PI3K derived more benefit from everolimus.Also, at 2013 ESMO  Conference, investigators presented data on patients from BOLERO-3 with low PTEN or high pS6 levels. Those inContrasting with these results, biomarker analysis from BOLERO-2, which involved 3,230 exons of 182 oncogenes and tumor-suppressor genes that were sequenced using next-generation sequencing, on 309 tissue samples, demonstrated a positive treatment effect in favor of everolimus across the various key genetic marker subgroups. In truthOther trials are currently being designed to specifically target the PI3K/AKT pathway in conjunction with HER2 itself, in order to hopefully achieve more durable and potent antitumor effects and overcome the resistant to treatment. These PI3K inhibitors are being introduced to the treatment of women with HER2+ BC in several stages of disease, from the neoadjuvant setting to the metastatic setting, after several lines of standard treatment.As an example, the NeoPHOEBE study (NCT01816594), recruited women with HER2+ newly diagnosed early BC larger than 2cm. Patients were randomized to receive neoadjuvant weekly paclitaxel and Trastuzumab in combination with an oral PI3K inhibitor, BKM120, or placebo. The study has been completed, and was conducted in two separate cohorts (PIK3CA mutated and PI3K3CA wild-type) using a two-stage approach. NCT02152943 is evaluating everolimus, letrozole, and trastuzumab.The combination of trastuzumab with a PI3K inhibitor was evaluated in a phase 1 clinical trial. Buparlisib plus trastuzumab was tested in a cohort of HER2+ metastatic BC patients resistant to trastuzumab-based therapy, with a manageable toxicity profile and preliminary evidence of clinical activity. Of note, inhibition of the PI3K/AKT/mTOR and RAS/MEK/ERK pathways was observed in paired tumor biopsies. Pan PI3KRecently, at ASCO 2015, an oral PI3Kα inhibitor, BYL719, was evaluated in combination with trastuzumab and a HER3 inhibitor, in a heavily pre treated HER2+ metastatic BC population (median number of previous therapy lines of 6). Clinical activity was observed in patients harbouring PIK3CA mutations. Nevertheless, gastrointestinal and metabolic toxicities were an important issue, warranting exploration of intermittent schedule regimens. The PIK3CA pathway may be targeted in other downstream points of the pathway, such as Akt. The Akt-inhibitor MK2206, was evaluated before surgery in patients with stage I, II or III BC with various combinations of ER, PR and HER2.Many other trials are being performing with the important aim to target the PI3K/AKT/mTOR pathway in conjunction with HER2. Toxicity associated with PI3K/AKT/mTOR pathway inhibitors may be taken into account, as well as the added financial cost. Deaths attributable to everolimus were seen BOLERO-1, due to pneumonitis, pulmonary embolism, respiratory failure, pulmonary edema, pneumonia, and cardiorespiratory arrest. Management of adverse events related to these drugs, especially when combined with chemotherapy, is of extreme importance.Therefore, the PI3K/AKT/mTOR pathway seems to play an important role in anti-HER2 therapy resistance. The optimal timing of targeting this pathway, either in the first line setting, or in a disease scenario where resistance is already established is yet to be determined. Also, the specific point of the pathway to be elected as the ideal target is still unclear. There are many options, ranging from mTOR-inhibitors to Akt-inhibitors and specific inhibitors of different isoforms of PI3K. Additionally, a balance between cost and side effects is of extreme importance in moving forward this strategy to clinical practice.The aminoterminal-truncated form of the HER2 receptor, that undergoes a proteolytic cleavage generating a truncated fragment, is denominated p95HER2 and is constitutively active. Also, p95HER2 fragments may arise through translation of the mRNA encoding HER2 from internal initiation codons. It has kThe presence of p95HER2 has been associated with worse survival outcomes in patients with localized HER2+ BC. Primary breast tumor tissues were evaluated by Western blot analysis for HER-2 protein forms, including p185HER-2 and p95HER-2. Authors found that a high level of p95HER-2 in primary tumor tissue correlated with reduced 5-year disease-free survival (DFS) . Differences in DFS were quite impressive between the two groups, namely 139 versus 32 months, for low and high levels of p95HER2, respectively.Despite being an independent prognostic factor in breast cancer, defining a group of patients with HER2+ disease with significantly worse survival outcomes, p95HER2 is also a marker of resistance to trastuzumab. Previous studies demonstrated that metastatic BC patients overexpressing p95HER2 had considerably lower response rates to trastuzumab compared with patients expressing full-length HER2. In this Other studies reported a correlation between elevated p95 expression and poor outcomes in response to trastuzumab treatment., 49In vitro experiments observed that chemotherapy sensitizes p95HER2/611CTF-positive patient-derived xenografts to trastuzumab, which may be related to HER2 stabilization induced by chemotherapy.[Interestingly, recent studies showed that tumors expressing the most active p95HER2 fragment, the so-called p95HER2/611CTF, indeed do respond to trastuzumab in combination with chemotherapy. The 611CTF fragment is highly oncogenic, because it spontaneously homodimerizes into a constitutively active form. In vitrootherapy. Another Larger studies are highly awaited for confirmation of p95HER2 as a biomarker of resistance or sensitivity to anti-HER2 therapy, enabling development of strategies to target this form of HER2. Evidence is accumulating indicating that trastuzumab alone is not effective in patients who have expression of p95HER2 isoforms. Figure Chemotherapy, in conjunction with trastuzumab, is essential to achieve response to therapy. Larger and well-controlled studies, using pre-defined clinical cutoffs for p95 expression and also blinded analyses are highly awaited.Insulin-like growth factor-I receptor (IGF-IR) is another pathway implicated in trastuzumab resistance. This recImportant data demonstrated that interaction between HER2 and IGFR-IR occurs in trastuzumab-resistant cells and that inhibition of IGF-IR tyrosine kinase activity may lead to decreased HER-2 phosphorylation with consequent restoration of trastuzumab sensitivity. This important cross talk between HER-2 and IGF-IR could potentially lead to clinical use of IGF-IR-targeted agents to block this receptor.In vitro studies demonstrated that inhibition of HER2 signaling using trastuzumab, and inhibition of IGF-IR signaling using a dominant negative construct produced synergistic growth inhibition of HER2-overexpressing breast cancer cells.[er cells., 56in vitro inhibition of IGF1R expression was made through small receptor tyrosine kinase inhibitors, response to trastuzumab was substantially improved, suggesting an interaction between HER2 and IGFR. [Of note, in experiments in which nd IGFR. Future clinical trials of HER2 and IGF1R-inhibitors are awaited to corroborate these preclinical findings. Analysis of potential predictive biomarkers to identify tumors that may achieve maximum benefit from this approach would be of great interest.c-MET is a receptor tyrosine kinase (RTK) encoded by the MET proto-oncogene that is largely expressed in epithelial/endothelial cells.  Upon binOverexpression of c-MET has been shown to contribute to the development of the invasive phenotype during BC progression. Poorly differentiated and invasive cell lines may express high levels of the receptor and ligation by HGF is associated with increased motility and invasiness.Previous trials have shown that overexpression of c-MET in BC primary tumors is strongly associated with worse disease-free survival compared to tumors without c-MET overexpression., 63MET expression might be associated with resistance to treatment in BC patients, even in a cohort of individuals co expressing ERBB2, in which a highly effective treatment, such as trastuzumab, is largely available.In fact, overexpression c-Met has emerged as a potential contributor to trastuzumab resistance and has been shown to be highly elevated in HER2-positive breast cancer cell lines and in 25% of HER2-positive breast cancer patients tissues., 65 LossCell line experiments demonstrated that foretinib, a multikinase MET inhibitor, combined with erlotinib or lapatinib, was capable of cell growth inhibition of MET-amplified or overexpressing cell lines. AdditionMany therapeutic strategies to target Met are currently under clinical development, including tyrosine kinase inhibitors and monoclonal antibodies, which could potentially be associated with anti-HER2 therapy in patients with BC overexpressing HER2 to optimize treatment and avoid tumor resistance. As an example, cabozantinib, a small molecule inhibitor of the tyrosine kinases c-Met and VEGFR2, is currently being evaluated in combination with trastuzumab, in HER2+ BC patients who have brain metastasis (NCT02260531).The proto-oncogene Src encodes for the non-receptor protein tyrosine kinase Src, which is involved in many cellular events, mediating cell proliferation and survival. de novo. In fact, data points to the fact that Src activation by itself is sufficient to confer trastuzumab resistance.[Src has extensive interaction with transmembrane receptor tyrosine kinases (RTKs) at the cell membrane, such as HER1 and HER2. Src activation confers resistance to trastuzumab, both acquired and sistance.In vitro targeting Src universally sensitized trastuzumab-resistant cells to trastuzumab treatment, and was able to suppress tumor growth in multiple preclinical resistance models. Other studies also demonstrated that Src activation is involved with trastuzumab mechanisms of resistance, and indicates poor prognosis in patients with HER2+ BC.[The relationship between SRC activation and patient response to trastuzumab-based therapies was previously studied in a cohort of 57 patients who were evaluated for SRC activation in primary breast tumors. As featured, patients with high amounts of phosphorylated Src (pSrc) in tumors demonstrated a lower clinical response rate and a higher progressive disease rate after trastuzumab therapy. Additionally, the OS of patients with high pSrc tumors (median 34.2 months) was significantly lower than that of patients with low pSrc tumors (median 57.9 months). In vitroHER2+ BC.Resistance to lapatinib was also demonstrated in Src activated cell lines. Saracatinib, a small molecule inhibitor of Src, was combined with lapatinib, and significantly prolonged survival of xenografted mice compared with saracatinib alone.Therefore, blocking Src interaction with HER2 is a promising strategy that might impact the management of HER2+ BC patients.Trastuzumab mechanism of action is dependent on the host immune system. As a consequence, trastuzumab is only partially effective in mice defective for the receptor of immunoglobulins (gamma receptor knockout mice). Innate and adaptive immune mechanisms are emerging as key players in modulation of the effects of HER2-targeted drugs.Therefore, sensitivity to anti-HER2 therapy depends on a well functioning host immune system. Loi et al. evaluated tumor PIK3CA mutations, lymphocyte infiltration, and RFS in an early HER2+ BC cohort of patients enrolled on the Finher Trial. , 73 In tIn the neoadjuvant setting, signals of immune system activation were associated with response to pertuzumab and trastuzumab in the Neosphere study. In this analysis, baseline core biopsies were collected from 387 out of 417 patients and evaluated by gene expression profiling. Patients identified as having low expression of PD-L1 had lower pathologic complete response rates (pCR). In contrast, individuals with high expression of STAT1 and IFNG, achieved high pCR rates, irrespective of estrogen receptor status.Tumor cells display genetic and epigenetic changes that provide plenty of tumor-associated antigens, which could potentially elicit host immune system recognition and tumor cell destruction. Avoidance of this host intrinsic defense mechanism is fundamental for tumor progression. As a consequence, immune-checkpoint proteins are frequently deregulated by tumors. CytotoxiAmong one of the first reports that recognized the importance of this rapidly evolving field in BC was presented at the 2013 SABCS. Breast tRecently, Perez et al. defined a group with enriched immune functions in patients enrolled in the N9831 trastuzumab adjuvant trial, based on the expression of 87 genes. Using geAnalysis of immune markers in HER2+ BC indicates that host immune system integrity is extremely important to trastuzumab response. Future clinical trials exploring patient immune system, with the ultimate goal of maximizing immune response, might result in great improvement in trastuzumab benefit and sensitivity.Table Lapatinib is a synthetic, orally active TKI that reversibly blocks phosphorylation of the epidermal growth factor receptor (EGFR), HER2, Erk-1 and-2 and AKT kinases . Other TLapatinib in combination with capecitabine is FDA-approved for the treatment of HER2+ metastatic BC that have received prior therapy with trastuzumab . Also, iin vitro synergistic interaction was demonstrated between those two drugs [Combining trastuzumab with lapatinib is an attractive strategy, since both intracellular and extracellular HER2 domains would be targeted. Indeed, wo drugs . Dual anwo drugs , and alswo drugs , 85. Nevwo drugs .Unfortunately, acquired resistance to lapatinib often develops in patients who initially responded to therapy. Chronic exposure to lapatinib may convert HER2-over-expressing BC cells that are initially sensitive to lapatinib-induced apoptosis to resistant cells . Importain vitro, suggesting that this is a strategy that might be a possible treatment option to be tested in future clinical trials [Small molecule TKIs are being studied in combination with drugs that target the PIK3CA/AKT pathway, which was previously discussed as being implicated in trastuzumab resistance. PI3K inhibitors in monotherapy might display modest clinical activity, and a potential resistance mechanism frequently involves increased expression and phosphorylation of the HER family of receptors, particularly HER2 and HER3 , 90. Thel trials .Another potential mechanism to lapatinib resistance is activation of AXL, which is a membrane-bound receptor tyrosine kinase . In a laThis humanized monoclonal antibody that binds to HER2 is an effective inhibitor of EGFR/HER2 driven signaling pathway. Compared to trastuzumab, much less is known about development of resistance to this targeted agent. Of note, in a tamoxifen-resistant BC cell line, pertuzumab promoted rapid formation of HER3/EGFR heterodimers, with subsequent phosphorylation of AKT and ERK1/2 . In thisIn ovarian cancer, microRNA-150 (miR-150) expression was induced by pertuzumab in a cell line model, and suppression of miRNA-150 resulted in decreased drug sensitivity to pertuzumab and cell apoptosis . miRNAs The development of resistance to anti-HER2 therapies seems complex, and is probably associated with multiple mechanisms. A generated transgenic mouse model of HER2+, PIK3CA mutant BC acquired resistance to trastuzumab, pertuzumab in combination with the pan-PI3K inhibitor BKM120 after treatment exposure. Initially, tumor regression occurred after 6 weeks of therapy. Nevertheless, within two months, all tumors relapsed. Of note, p95 HER2, which was not detected in untreated tumors, was expressed in resistant clones, and HER2 expression was significantly reduced . This daTrastuzumab emtansine (T-DM1) is an antibody-drug conjugate that is active in second line, as well as later lines in the treatment of advanced HER2+ BC that has progressed on trastuzumab therapy , 100. AlPrimary resistance to T-DM1 may be relatively infrequent, particularly in patients who have no prior exposure to trastuzumab. The most common scenario is found in patients who initially responded, but eventually ceased to respond, despite continued treatment with T-DM1. It is important to know that DM1 and its metabolites need to accumulate in cell cytoplasm to reach a concentration that exceeds the threshold needed to evoke cell death . ConsequAnother important resistance mechanism of T-DM1 is mediated by neuregulin b1 (NRG) , the ligOther HER2-targeting antibody-drug conjugate (ADC) are currently under development, such as SYD985. In the ongoing phase 1 clinical trial, safety and efficacy of SYD985 have been evaluated in European Oncology Centers, where patients with locally advanced or metastatic solid tumors of any origin were treated with SYD985. Of note, objective responses were seen in metastatic HER2+ BC patients who were refractory to previous HER2-targeted treatments, including T-DM1 .de novo. In either case, this is a potentially life-threatening event, which may lead to rapidly progressive disease, or can result in important quality of life impairment for patients, due to disease related symptoms. A better knowledge of these mechanisms is of extreme importance for the development of effective strategies to overcome resistance.Resistance to trastuzumab and other anti-HER2 therapies is an event that may occur during the course of therapy or In developing more effective and durable treatment strategies, multitargeted therapy should be considered. The recognition of specific molecular predictors of response to emerging therapies will allow a more personalized approach to the treatment of HER2-amplified BC.Enrolling patients into clinical trials, with the purpose of understand and target the molecular mechanisms involved in HER2 therapy resistance is of crucial importance. Laboratory, pre clinical and clinical findings, such as seen at the BOLERO-1 and 3 trials, are very promising in this field, but have not yet been incorporated in clinical practice. Also, a better characterization between the relationship of tumor inflammatory infiltrate or molecular inflammatory signature, and trastuzumab benefit is warranted, before its incorporation in clinical practice.Nowadays, the focus on HER2 expression/amplification status alone is not a realistic approach to understand the underlying mechanisms of disease progression and resistance. Improvement of patient outcomes in this disease setting will only be clinically meaningful if resistance pathways involved in disease progression are better recognized, understood and targeted.


HER2)‐positive breast cancer. For dichotomous variables, the relative risk ratio (RR) and 95% confidence interval (CI) were used to investigate outcome measures: pathological complete response (pCR), neutropenia, diarrhea, dermatologic toxicity, and congestive heart failure (CHF). Eight randomized controlled trials of 2350 participants  were selected to compare the efficiency and safety of lapatinib to trastuzumab. A significant difference was found between lapatinib and trastuzumab for pCR . In six studies, a significant difference was found between trastuzumab and combination therapy for pCR , diarrhea , and dermatologic toxicity , but none was found for neutropenia  or CHF . Combination therapy compared to trastuzumab alone increases the pCR rate of HER2‐positive breast cancer patients with no additional cardiac events. Trastuzumab, which is still the first‐line therapy in breast cancer, increases the pCR rate more than lapatinib.This meta‐analysis compared the efficiency and safety of lapatinib and trastuzumab, alone or in combination, administered with neoadjuvant chemotherapy in patients with human epidermal growth factor receptor 2 ( Human epidermal growth factor receptor 2 (HER2) is a member of the ErbB family of receptors and is overexpressed in 15–20% of breast cancer. Considered a prognostic and predictive marker of breast cancer, HER2 is highly aggressive and significantly reduces disease‐free and overall survival Two therapeutic approaches are used to inhibit HER2‐mediated signaling. The first approach is trastuzumab, a humanized monoclonal antibody that blocks the activity of HER2, which improves the prognosis of early and advanced stage HER2‐positive breast cancer A meta‐analysis of all relevant published randomized, controlled trials (RCTs) was performed to compare the efficiency and safety of lapatinib and trastuzumab, alone or in combination, administered with neoadjuvant chemotherapy in patients with HER2‐positive breast cancer.The search strategy consisted of a systematic review of the literature for RCTs over the last 5 years in any language in the Wanfang Data, PubMed, the Cochrane Library, Medline, and EBSCO databases. The publication time searched was from March 2011 to March 2016. The search terms used were “trastuzumab,” “lapatinib,” “her‐2 positive,” “breast AND (cancer OR tumor OR carcinoma),” and “randomized controlled trials OR RCT.” Article selection was based on the methodology used in the RCTs. A total of 39 Chinese articles and 506 English articles were identified. These articles were reviewed and screened for duplicate or incomplete data. When relevant data were unclear, the articles were read by different investigators and then discussed.Inclusion criteria were RCTs that (1) pathologically confirmed breast cancer and HER2 positivity, (2) patients aged over 18 years, (3) chemotherapy tolerance, (4) expected lifetime of more than 3 months, (5) compared trastuzumab with lapatinib or trastuzumab versus the combination therapy, and (6) reported sufficient data on outcomes. Exclusion criteria were (1) nonrandomized and nonclinical controlled trials, (2) trials with missing data, and (3) duplicate reports, trials of poor methodological quality and trials with obvious bias.Data extracted from each article included the name of the first author, year of publication, journal name, study quality, intervention, number of patients in the study, dosage used in the three groups , and the number of patients with different endpoints.Several outcomes were measured: pCR, neutropenia, diarrhea, dermatologic toxicity, and congestive heart failure (CHF). pCR was defined as the absence of any invasive component in the resected breast specimen. Adverse events were graded according to the National Cancer Institute Common Toxicity Criteria Version 3.0. The number of patients with grade 3–4 adverse events was determined from the articles. Disagreement regarding data extraction was resolved by discussion and consensus among the investigators.The methodological quality of the RCTs was assed according to the Cochrane Handbook for Systematic Reviews of Interventions Version 5.0.0: (1) random sequence generation, (2) concealment of allocation, (3) blinding of participants and personnel, (4) blinding of the outcome assessment, (5) incomplete outcome data, (6) selective reporting, and (7) other sources of bias.P‐value of <0.05 was considered statistically significant. The inconsistency index (I2) statistic and the Q statistic were used to test the heterogeneity between RCTs P > 0.1; I2 ≤ 50%), a fixed effect model was used for secondary analysis; otherwise , a random‐effect model was used Data were analyzed using Review Manager v.5.3 software . For dichotomous variables, outcomes were calculated as the relative risk ratio (RR), the 95% confidence interval (CI), and a A total of 39 Chinese papers and 506 English papers were selected, published from March 2011 to March 2016. The literature selection process is presented in the PRISMA flowchart . The heterogeneity test was not statistically significant; therefore, data for each outcome was calculated using the fixed effects model . The meta‐analysis indicated a significant difference in the pCR rate in patients treated with trastuzumab compared to lapatinib   were also analyzed. Compared to trastuzumab alone, combination therapy showed a higher pCR rate   Fig. . Six stuI2 = 68%; P = 0.009). The six studies included in the meta‐analysis indicated no significant difference in the neutropenia adverse event between the lapatinib and trastuzumab group  . The four studies included in the meta‐analysis indicated no significant difference in the neutropenia adverse event in patients between the trastuzumab and combination groups   Fig. .I2 = 0%; P = 0.81). The seven studies included in the meta‐analysis indicated a significant difference in the diarrhea adverse event in patients between the lapatinib and trastuzumab groups  . The four studies included in the meta‐analysis indicated a significant difference in the diarrhea adverse event in patients between the trastuzumab and combination groups  . The seven studies included in the meta‐analysis indicated a significant difference in the dermatologic toxicity in patients between the lapatinib and trastuzumab groups  . The five studies included in the meta‐analysis indicated a significant difference in the dermatologic toxicity in patients between the trastuzumab and combination groups  . The five studies included in the meta‐analysis indicated no significant difference in the CHF in patients between the lapatinib and trastuzumab groups    Fig. .This study compared the efficiency and safety of lapatinib and trastuzumab, alone or in combination, combined with neoadjuvant chemotherapy in patients with HER2‐positive breast cancer. The meta‐analysis provided evidence that lapatinib and trastuzumab combination therapy significantly increased the pCR rate. Trastuzumab increased the pCR rate more than lapatinib. Lapatinib caused skin rash, diarrhea, and other adverse events.Neoadjuvant chemotherapy has been reported to be equivalent to adjuvant chemotherapy in terms of survival and overall disease progression in the treatment of breast cancer Trastuzumab substantially improves the efficacy of chemotherapy in HER2‐positive breast cancer patients. Resistance to trastuzumab still develops in some women after adjuvant therapy, which increases the importance of the development of additional agents that target HER2 through different mechanisms of action P = 0.003). Moreover, the pCR rate of the combination group was 1.33 times higher than that of the trastuzumab group. These results might be explained by the lower ability of lapatinib to block the HER2 pathway compared to trastuzumab. Furthermore, trastuzumab may have additional antitumor efficacy through the recruitment of immune effector cells responsible for antibody‐dependent cytotoxicity and inhibiting angiogenesis In the eight RCTs, the pCR rate as the first endpoint was analyzed for the lapatinib, trastuzumab, and combination groups. A significant difference in the pCR rate between the lapatinib and trastuzumab groups was found P = 0.00. MoreoveThe meta‐analysis analyzed grade 3–4 adverse events according to the national criteria. Common adverse reactions of lapatinib were diarrhea and skin rash while trastuzumab showed potentially serious cardiac toxicity. The incidence of adverse events, such as diarrhea and skin rash, in the lapatinib group was significantly higher than in the trastuzumab group. Lapatinib and trastuzumab combination therapy also exhibited the same effect. Surprisingly, the results demonstrated that the combination did not increase cardiac toxicity compared to trastuzumab, which indicates that combination therapy is safe and effective.This meta‐analysis had several limitations. As shown in Table In conclusion, the currently available evidence shows that lapatinib and trastuzumab combination therapy significantly increases pCR rates in HER2‐positive breast cancer patients with no additional cardiac side effects compared to trastuzumab alone. Trastuzumab is better than lapatinib according to the pCR rate from our data. However, trastuzumab is still the first‐line targeted therapy in breast cancer and cannot be replaced by lapatinib because of cost considerations. Large, high‐quality, double‐blind trials are needed to confirm the efficiency of lapatinib and trastuzumab combination therapy, and verify whether combination therapy can prolong disease‐free survival or overall survival.The authors declare that they have no conflict of interest.


Betula pendula. Broad-sense heritability (H2) and coefficient of genotypic variation (CVG) were estimated for metabolites in senescent leaves and litter using 19 genotypes selected from a B. pendula population in southern Finland. We found that most of the secondary metabolites remained through senescence and decomposition and that their persistence was related to their chemical properties. Intrapopulation H2 and CVG for intracellular phenolics, epicuticular flavonoid aglycones and condensed tannins were high and remarkably, increased from senescent leaves to decomposed litter. The rank of genotypes in metabolite concentrations was persistent through litter decomposition. Lignin was an exception, however, with a diminishing genotypic variation during decomposition, and the concentrations of lignin and condensed tannins had a negative genotypic correlation in the senescent leaves. Our results show that secondary metabolites and their intrapopulation genotypic variation can for the most part remain through leaf senescence and early decomposition, which is a prerequisite for initial litter quality to predict variation in litter decomposition rates. Persistent genotypic variation also opens an avenue for selection to impact litter decomposition in B. pendula populations through acting on their green foliage secondary chemistry. The negative genotypic correlations and diminishing heritability of lignin concentrations may, however, counteract this process.Abundant secondary metabolites, such as condensed tannins, and their interpopulation genotypic variation can remain through plant leaf senescence and affect litter decomposition. Whether the intrapopulation genotypic variation of a more diverse assortment of secondary metabolites equally persists through leaf senescence and litter decomposition is not well understood. We analyzed concentrations of intracellular phenolics, epicuticular flavonoid aglycones, epicuticular triterpenoids, condensed tannins, and lignin in green leaves, senescent leaves and partly decomposed litter of silver birch,  Plants produce an abundance of diverse secondary metabolites such as phenolics and terpenoids. These compounds were thought to be waste products until ortho-diphenol) are preferential substrates for PPOs  remain and preserve their genotypic variation through the early phase of litter decomposition. These hypotheses, if supported, would manifest the persistence of genotypic variation of foliar chemistry through senescence and decomposition: a prerequisite for the initial litter quality to predict and the selection acting on foliar chemistry to affect litter decomposition.In this study, we focus on the fate of foliar secondary metabolites and the persistence of their genotypic variation through leaf senescence and litter decomposition in a n Europe , where ie growth  and physe growth  as well e growth  and decoe growth , 2015. Fe growth , their ge growth  are equar larvae . The abile found , but theB. pendula trees . The trees that grow in Kuikanniitty consist of the micropropagated progeny of 30 la trees , selectela trees . The soun = 5) on June 26. The collected leaves were immediately frozen in liquid nitrogen. For collecting senescent leaves, two south-side branches (at the height of 140–300 cm) of each tree were enclosed in mesh bags before leaf fall (September 8 to 10) in all six blocks . The mesh bags were collected after leaf fall (October 28 to 30), their contents were pooled within a tree and random subsamples of leaves were taken for laboratory analyses. Remaining leaves were stored in plastic bags in 4°C until November 5, when 10-g  samples were used to establish litter patches on the ground of a clear-cut, B. pendula-Pinus sylvestris forest site in Loppi, south Finland . The soil in this site is post-glacial sorted fine sand with a pH of 5.0 and total C and N concentrations of 6 and 0.3%, respectively, in the upper 0–5 cm layer (Pteridium aquilinum (L.) Kuhn, grasses Calamagrostis arundinacea (L.) Roth and Deschampsia flexuosa (L.) Trin., and dwarf shrubs Vaccinium myrtillus L. and Vaccinium vitis-idea L.  was collected from the trees in five replicate blocks , and again extracted with 1 ml of 100% for 10 min. The supernatants were dried in a vacuum concentrator at 45°C and stored at 4°C.3+ -reagent were added and the suspension was incubated at 90°C for 50 min. After being cooled with ice, the absorbance of the suspension was measured at 550 nm using cyanidin chloride  as a quantification standard.Lignin was determined from the precipitants  using the method described in 2O and (B) 0.1 % formic acid in acetonitrile . The flow was 0.41 ml min-1 and the elution was performed with a gradient as follows: B started with 5%, was increased to 50% by 15 min, to 60% by 35 min, to 85% by 45 min and to 98% by 50 min and then kept at 98% for 5 min. The column was returned to its starting condition with 15 min equilibration, giving a total of 70 min for each run. The injection volume was 12 μl with a partial loop. The MS was run in a positive ion mode with a mass range of 150–1500 m/z. The capillary temperature was kept at 320°C and the voltage at 5 V. The sheath gas flow rate was kept at 20 ml min-1, auxiliary gas flow rate at 5 ml min-1 and sweep gas flow rate at 5 ml min-1. The tube lens was set to 80 V. The areas for the compounds were integrated using the Xcalibur software.For quantifying the concentrations of small-molecular phenolics and triterpenoids, a 500-μl aliquot of the methanol resuspension was dried in a vacuum centrifuge at 45°C and stored at –20°C. After storage, the samples were dissolved in 250 μl 100% methanol and 250 μl distilled water. High-performance liquid chromatography-mass spectrometry (HPLC-MS) was then performed using Thermo Finnigan LC with the flow split into two between a Thermo LTQ MS  with electrospray ionization (ESI) and a Finnigan PDA detector with a subsequent Corona Ultra charged aerosol detector. The column was C-18 Luna with an inner diameter of 2 mm, length of 150 mm and a particle size of 3 μm . The temperature of the tray was set to 18°C and the column to 40°C. The solvents were (A) 0.1% formic acid  in HD-glucopyranoside (DHPPG); (+)-catechin (Aldrich) for (+)-catechin; quercetin 3-glucoside (Extrasynthese) for myricetin, quercetin, and kaempferol derivatives; and acacetin (Extrasynthese) for flavonoid aglycones. Triterpenoids are reported as arbitrary units (peak area g-1 dry mass). For the analyses of heritability and statistical significance of genotypic variation in the senescent leaves and decomposed litter, the small-molecular phenolics and triterpenoids were grouped into intracellular phenolics -catechin, myricetin glycosides, quercetin glycosides, and kaempferol glycosides), epicuticular flavonoid aglycones and epicuticular triterpenoids. In addition, the intracellular phenolics were tested as subgroups; i.e., phenolic acids (CQAs and CouQAs), myricetin glycosides, quercetin glycosides, and kaempferol glycosides.The compounds were annotated using retention times, UV spectra and HPLC-MS. Peak picking was done using MetAlign software  based onTo avoid a multitude of mean tests and to allow easy statistical inference in the graphs , we inteB. pendula genotypes, all trees within a genotype had an equal genetic structure. In such material, all variation that is found within genotypes can be considered to be due to the variation in environment, or due to a measurement error, and all variation found between the genotypes to be genetic (H2) can be calculated  were calculated according to the Eq. 2, where  is the phenotypic mean.Coefficients of genotypic variation . Compounds with qualitative variation were excluded from these analyses and the senescent leaves and decomposed litter were analyzed separately as not all the compounds of senescent leaves were present in litter. Before PCA, both columns and rows of the data matrix were transformed to have a mean of zero and a standard deviation of 1. This was done to reduce the quantitative differences among the compounds and the samples and thus, focus more on chemical profiles. Compounds with skewed distribution were logGenotypic correlations among the compound groups and between the senescent leaves and decomposed litter were tested using the Spearman rank correlation analysis. The persistence of genotypic variation in the chemical profiles revealed by PCA between the senescent leaves and litter was tested as rank correlations of the genotype means of PC axis scores. All statistical analyses were performed using the SPSS 15.0.1 and SPSS 18 statistical packages  except for the PCA, which was performed using the SIMCA-P+ software .Figure 1A). The concentrations of CouQAs were on average 97% lower in the senescent than green leaves and decreased below the detection limit during litter decomposition . DHPPG concentration was 99% lower in the senescent than green leaves and also decreased below detection during decomposition .All those secondary metabolites that were found in green summer leaves were also detected in senescent leaves, except for CQAs . Concentrations of myricetin glycosides decreased on average by 93% during leaf senescence and none of the six compounds was detected in the decomposed litter . In contrast, concentrations of quercetin and kaempferol glycosides decreased on average by 79 and 76% during leaf senescence and all compounds, except for kaempferol-3-glucoside, were also detected in the decomposed litter . Based on the comparison of confidence intervals, the reduction in concentration during leaf senescence was statistically significant for all flavonol glycosides, except for kaempferol 3-arabinofuranoside . During litter decomposition, the concentration of quercetin and kaempferol glycosides decreased on average by 86 and 52%, and except for kaempferol 3-glucuronide, the decrease was statistically significant in all compounds .The decrease of flavonol glycoside concentrations during leaf senescence and litter decomposition varied among the flavonoid subgroups . The concentrations were on average 27% lower in the senescent than green leaves and the decrease was statistically significant for nine of the 15 compounds . For one of the compounds (F15), the concentration increased by 70% . During litter decomposition, the concentrations of flavonoid aglycones decreased on average by 51% and the decrease was statistically significant for all compounds .The concentrations of epicuticular flavonoid aglycones varied a lot in the green leaves, but displayed relatively similar dynamics during leaf senescence and litter decomposition , but this decrease was driven by one abundant compound T8 that was annotated as 12-O-acetyl-3-O-malonylbetulafolientriol. When T8 was excluded from calculations, the mean concentration of triterpenoids increased by 4%, and for the two ocotillol-type triterpenoids, papyriferic acid (T7) and its derivative (T6), the increase was statistically significant . During litter decomposition, all triterpenoids had parallel dynamics, the mean concentration decreased by 55% and the decrease was statistically significant for all compounds .The mean concentration of triterpenoids decreased during leaf senescence by 25% . During litter decomposition, lignin concentration increased further by 23%, but tannin concentration decreased by 87% . The (+)-catechin concentration decreased by 97% during senescence, but did not change during decomposition .Of the polymers, the concentration of lignin increased by 51%, while the concentration of condensed tannins did not change during leaf senescence .The secondary metabolites displayed both qualitative (absence or presence in only certain genotypes) and quantitative  genotypic variation. Qualitative variation was found among flavonol glycosides: the 3-glucuronides were lacking in four of the 19 genotypes  and the 3-arabinofuranosides were found in four genotypes only . The qualitative variation remained through the senescence and decomposition as these compounds were also found in the decomposed litter . Of the main metabolite groups, the epicuticular triterpenoids  had the highest broad-sense heritability, H2 (0.281) and coefficient of genotypic variation, CVG (0.138) in the senescent leaves (Table 1). The other groups had very similar heritabilities (0.113–0.121), whereas the CVG varied more, with lignin and epicuticular flavonoid aglycones having lower CVG than intracellular phenolics and condensed tannins (Table 1). The genotypic variation was statistically not highly significant in the senescent leaves, except for triterpenoids (Table 1). Among the intracellular phenolic subgroups, myricetin glycosides and kaempferol glycosides had very high values of H2  and CVG (0.331 and 0.219), while those of phenolic acids and quercetin glycosides resembled the values of intracellular phenolics in general . As a result, the decomposed litter had statistically highly significant genotypic variation in all compounds except for lignin that had lost genotypic variation during decomposition (Table 1).Quantitative genotypic variation was found in all compound groups  and PC2 mostly genotypic variation  . In the decomposed litter, genotypic variation was significant along both the PC1  and the PC2  . The ranks of genotype mean scores correlated positively between the senescent leaves and decomposed litter for PC2 , but not for PC1 . The genotype 16 was most distinct from others in both senescent leaves and decomposed litter . Triterpenoids and the most lipophilic flavonoid aglycones (F11-F15) were the compounds that best explained the genotypic variation along the PC axes . In the decomposed litter, four triterpenoids, including papyriferic acid (T7) and its derivative (T6), formed a tight cluster separated from the rest of the compounds .The genotypic variation in the small-molecular compounds of senescent leaves was also clearly visible in the PCA of individual compounds, where PC1 represents environmental variation . Among the intracellular phenolic subgroups of the senescent leaves, concentrations of phenolic acids correlated positively with the concentrations of quercetin glycosides  and kaempferol glycosides , which also correlated with each other . The ranks of genotype mean concentrations correlated positively between the senescent leaves and decomposed litter for intracellular phenolics , flavonoid aglycones , triterpenoids  and condensed tannins , but not for lignin  .The genotype mean concentrations of the two epicuticular compound groups – flavonoid aglycones and triterpenoids – were positively correlated in the senescent leaves, whereas the mean concentrations of condensed tannins correlated negatively with the concentrations of flavonoid aglycones and lignin  remained in the senescent leaves. As we hypothesized, the remaining metabolites also exhibited significant genotypic variation with high broad-sense heritabilities and coefficients of genotypic variation. During decomposition, most metabolites decreased in concentration, suggesting that they were decomposed faster than the litter material on average, but the genotypic variation was persistent. This was manifested by the increasing heritabilities and coefficients of genotypic variation of the intracellular phenolics, surface flavonoid aglycones and condensed tannins during litter decomposition. Confirming the persistence of genotypic variation, the genotype ranks in metabolite concentrations remained stable under field conditions and microbial degradation. Considering that secondary metabolites can affect litter decomposition and nutrient cycling  moiety, which makes them preferential substrates for PPOs (The absence of CQAs in the senescent leaves was expected due to their catechol (for PPOs . Monophefor PPOs .-1 dry mass) in the green leaves, but lost little of their concentration during leaf senescence. Epicuticular flavonoids are typically highly methylated, which makes them lipophilic, but at the same time shields the reactive hydroxyl groups from oxidation . The two triterpenoids that increased in concentration during senescence were both ocotillol-type of oxidized dammaranes . These are known to be formed from corresponding dammaranes in oxidative conditions, e.g., through UV-induced photochemical reactions  during the senescence. In the PCA of decomposed litter, two other triterpenoids (T1 and T3) clustered together with the oxidized ocotillol-type triterpenoids (T6 and T7), which suggests that the concentration pattern of these compounds was also influenced by oxidative processes.The flavonoid aglycones, which situate on leaf surface , had a lxidation . During er input . The otheactions . It is tB. pubescens ssp. czerepanovii  of growth has earlier been estimated to vary between 0.10 and 0.19 for those trees, from which we collected the litter  that have high papyriferic acid production. Recent results also suggest that the concentrations of condensed tannins in leaf litter could be the target of selection due to the beneficial effects of high tannin concentrations on plant nitrogen uptake in certain conditions  and initial litter quality hypothesis (most other metabolites) , which is known to defend birch twigs against hare browsing , 1996, wtes) cf. . The inites) cf. . Our restes) cf.  and inhites) cf. , and sintes) cf. .B. pendula. Epicuticular flavonoid aglycones and triterpenoids are synthesized in multicellular peltate glands on birch leaves and twigs and excreted to the surface as a mixed resin to provide chemical defense and protect from desiccation , which is a prerequisite for a long-term genetic influence on decomposition. However, we also found significant genotypic correlations among the compound groups. Of these, the positive correlation between flavonoid aglycones and triterpenoids has earlier been found in the green leaves  and barkiccation . By contB. pendula green foliage remain in the senescent leaves, although in much lower concentrations, and can withstand the first phase of decomposition. The persistence of the compounds appears to be related to their chemical properties, and particularly to their susceptibility to oxidation. It further appears that the genotypic variation of metabolite concentrations persists through leaf senescence and litter decomposition. This supports the hypothesis that initial litter quality can explain the variation in litter decomposition rates. It also opens an avenue for selection to impact litter decomposition in B. pendula populations through acting on their green foliage secondary chemistry. Whether this takes place, however, likely depends on the relative strength of the link of litter decomposition to secondary metabolites in comparison to the other attributes of B. pendula litter. At a particular forest site, finally, the significance of changes in litter metabolite concentrations caused by the selection apparently depends on the relative importance of litter chemistry among other biotic and environmental factors that control litter decomposition rates.The green foliage secondary chemistry of a tree population is a reflection of various selection forces, such as herbivory, which act on the genotypic structure of the population. Our results show that a majority of those secondary metabolites that have earlier been found to characterize JM designed the study and MR arranged the plant material; UP and HK carried out the field and laboratory work; TS contributed to the field work and SK-S and MK guided the laboratory analyses; UP, SK-S, and MK interpreted the data; JM, UP, SK-S, and TS analyzed the data; UP and JM wrote the manuscript and SK-S, MK, MR, and TS developed the text.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


It has come to our attention that Dr. Sreenivas Gannavaram does not meet criteria necessary to be listed as an author, as specified by Frontiers; therefore, he has been removed from the authors' list and mentioned Acknowledgments instead.The updated Author Contributions statement and Acknowledgments are below.The authors apologize for this error and state that it does not change the scientific conclusions of the article in any way.The original article has been updated.NT, VK, MG, and AS: designed and performed the experiments and co-wrote the MS. SS and VS: assisted in design of the study, statistical analysis, and MS writing. RS: conceived, designed, directed, and supervised the complete study.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Phenylketonuria (PKU) is an autosomal recessive genetic disease, caused by the phenylalanine hydroxylase (PAH) deficiency in the metabolic pathway, which prevents phenylalanine from being converted into tyrosine, leading to a large amount of phenylalanine discharged from the urine. Therefore, it is necessary to establish a simple, fast, accurate and reliable PKU molecular diagnostic method for clinical diagnosis.We established a novel diagnostic method by combining a single-tube multiplex PCR technique with molecular hybridization technique. The method was verified by DNA sequencing technology. The established new technology successfully detected 9 common PAH gene mutations in the Chinese population.Double-blind analysis indicated that the diagnostic accuracy and specificity of the PKU sample were all 100%. Frequencies of single mutation R111X, R176X, Ex6–96A, R241C, R243Q, R252Q, Y356X, V399 V and R413P genotypes were 8, 0.5, 16.5, 1.5, 27, 4.5, 13, 10.5, 8.5% respectively.The established method of combing single-tube multiplex PCR with molecular hybridization technology can accurately and rapidly detect PAH gene mutations in Chinese and is suitable for screening of large PKU populations with clinical samples. Phenylalanine hydroxylase (PAH) is an enzyme which is necessary to metabolize the amino phenylalanine to the amino acid tyrosine. It is a key enzyme in the phenylalanine metabolic pathway . PhenylkPAH gene is located on chromosome 12q22-q24.1 and contains 13 exons and 12 introns encoding with a protein containing 452 amino acids. At present, more than 600 mutations of PAH have been reported worldwide, and more than 70 mutations have been detected in China, mostly on exon 6th, 7th, 11th, and 12th exon. Among them, the 9 mutations R111X, R176X, Ex6–96A > G, R241C, R243Q, R252Q, Y356X, V399 V, R413P are the most common types of PAH mutations in Chinese population [PAH gene in different ethnicities and different regions are quite different and show great genetic heterogeneity, therefore, rapid detection of race-specific and common mutations is the focus of research. As a mature technology, reverse dot blot (RDB) technology has been successfully applied to the genotypic diseases such as thalassemia and Glucose-6-phosphate dehydrogenase(G6PD) deficiency [The ficiency . In thisA clinically tested blood sample was drawn and anticoagulated using EDTA-2Na-. The DNA was extracted using the Qiagen Whole Blood Genomic DNA Extraction Kit . Thirty DNA standard samples of 30 PAH genotypes identified by DNA sequencing were 27 in 9 mutants and 3 in wild-type. The R243Q (G → A) genotype was an artificial plasmid used for the establishment of this method. .PAH gene mutations detected include the following sites: R111X (c.331C → T), R176X (c.526C → T), Ex6–96A > G (c.611A → G), and R241C (c.721C → T).), R243Q (c.728G → A), R252Q (c.755G → A), Y356X (c.1068C → A), V399 V (c.1197A → T), R413P (c.1238G → C). In this study, five pairs of primers were designed to amplify the 9 mutation site fragments. A total of 18 probes were designed, of which 9 probes were used to detect the mutation sites and the other 9 probes were normal control probes. The 5′ end of the primer was biotin labeled.Primer 5.0 software was utilized to design the corresponding primers and detection probes. The PAH wild-type gene and mutant detection probes both crossed the point mutation site, and their 5’ends were aminated (5′-NH2). The sequence of primers and probes is illustrated in Tables The designed ted 5′-NH. The seqIn this study, biotinylated PCR products were hybridized with oligonucleotide probes on the solid phase to achieve PAH genotyping.3 -Na2CO3 buffer (pH 8.0) at the corresponding position. Dried in the air for 20 min, and then soaked in a nylon membrane with 0.1 mol/L NaOH for 10 min. After rinsing with distilled water, dry at room temperature for 10 min.The molecular hybrid membrane was prepared as follows: The carboxyl nylon membrane  was acidified with 0.1 mol/L HCl for 10 min, and the nylon membrane was activated with ethyldimethylaminopropyl diimine  for 10 min. Rinsing the membrane with distilled water twice and air dried for 20 min. Then, according to the layout of the probe on Fig. The PCR conditions were as follows: In a 25 μL PCR reaction system, 1 μL PCR buffer, 400 μmol/L dN(U)TP, and 0.2 μmol/L PAH primers, and 2 U Taq enzyme  and 0.5 U UNG enzyme  were used. The PCR reaction was performed on a T100 PCR machine . The reaction conditions were incubation at 50 °C for 15 min, predenaturation at 95 °C for 10 min, then 95 °C, 30s; 57 °C, 30s; 72 °C, 30s for 40 cycles, and 72 °C for 5 min.After the amplification was completed, 5 μL of the PCR product and 5 μL of DL2000 marker  were electrophoresed on a 1.5% agarose gel. Observe the electrophoresis result on a gel imager .Molecular hybridization assays include hybridization, washing, incubation, washing, and color development. The hybridization solution used for molecular hybridization analysis is presented in Table Hybridization: a membrane strip was placed in a hybridization tube containing 10 mL of Liquid A. Single-tube multiplex PCR amplified products were added. After treatment at 100 °C for 10 min, hybridization was performed in a molecular hybridization oven  at 45 °C for 2 h. At the same time, added about 45 mL of Liquid B to a 50 mL centrifuge tube and placed in a hybridization chamber for preheating.Washing the membrane: After the hybridization is completed, the membrane strip was moved to the liquid B and the membrane was washed at 45 °C for 10 min.Incubation: Prepare the incubating liquid and incubate the membrane strip in it for 30 min at room temperature.Washing the membrane: Discard the incubating liquid, add Liquid A and gently wash the membrane twice at room temperature for 5 min each time. After draining, add Liquid C and wash the membrane for 2 min at room temperature.Color development: Prepare a Color-substrate solution, soaked the membrane strip in it and protected from light for 10 min. After the color development is completed, wash the membrane with deionized water once to observe the result.It is judged by whether a blue spot signal appears at the test site or not. It’s indicating that the sample is wild type when all normal sites (N) on the membrane were developed and all mutation sites (M) were not colored. On the contrary, Mutation sites (M) appeared on the strip indicates that the site is positive.Ten DNA samples of the 10 (including wild-type) PAH genotypes mentioned above were taken and replicated within the batch (3 times) and inter-assay (10 days) tests to determine the repeatability of the PCR-Reverse Dot Blot method. One hundred eighty PKU-deficient and 20 PKU-normal DNA samples were tested by DNA sequencing and the established method in the double-blind experiment to assess specificity and accuracy.Using multiplex PCR in a single tube, we amplified PAH gene fragments of the detection loci whose size is 338 bp, 274 bp, 232 bp, 191 bp, and 145 bp respectively. The electrophoresis band is clear and without smear, indicating that the established PCR system can efficiently amplify PAH gene fragments of blood DNA  using the method above were validated by DNA sequencing analysis. The results of PCR-Reverse Dot Blot are consistent with DNA sequencing analysis results, it’s shown that the detection method, PCR amplification combined with reverse dot hybridization technique, established in this study can be used to detect common mutants of PAH.The repetitive test results showed that hybridized spots have a consistent color depth detected in wild-type and mutation PAH samples by the above method.As for the result of the inter-batch repeat experiment, due to different room temperature, it has a certain degree of color depth difference but it does not affect the interpretation of results. Before methodological comparison, the designed probes and primers should be validated in multiple samples to ensure that the color of each point is uniform and visible to the naked eye.The specificity and accuracy of the PCR-RDB technique were evaluated using 200 DNA samples of known PAH genotypes. A double-blind controlled trial was conducted so that neither the researchers nor the participants knew who they had taken. And the results of the double-blind controlled trial show that the genotypes of all DNA samples detected by this technique were identical to the results of the DNA sequencing analysis, with 100% specificity and accuracy.In this study, the frequencies of different genotypes were shown in Table DNA sequencing results of partial samples were shown in Fig. 4), so PKU is divided into typical PKU and BH4 deficient PKU, the former accounted for 98 to 99% [4 PKU patients use BH4 therapy [Phenylketonuria is an amino acid metabolic disease that is mainly caused by a decrease or absence of PAH activity in the human body. The function of PAH requires the use of tetrahydrobiopterin , PCR-single strand conformation polymorphism(PCR-SSCP), denaturing high-performance liquid chromatography (DHPLC), direct sequencing and other techniques are mainly applied to detect PAH gene mutations in China –14. The PAH gene in the Chinese population. The experimental results are in full compliance with the sequencing results. This method is characterized by good repeatability, high specificity, and high accuracy. It can detect 9 common PAH gene mutations in the Chinese population in one PCR reaction and one molecular hybridization within one working day. The method is cheap and convenient. This research provides a simple, rapid and accurate gene detection method for clinical screening of large populations of PAH gene mutations.In this study, the RDB technique was used to detect 9 common mutation- sites in the


The objective of the current study was to characterize the relationship between diet quality and body composition in participants living with IBD, specifically Crohn’s disease (CD) or ulcerative colitis (UC), in Atlantic Canada. Participants from the Atlantic Partnership for Tomorrow’s Health (PATH) study are residents of one of the four Atlantic Canada provinces. Participants who completed the dietary questionnaire and had body composition measured were included in the study . A greater number of participants with IBD reported having multiple chronic conditions compared to those without IBD. Those with UC had statistically higher body weight and body mass index (BMI) compared to those without IBD. Overall, significant positive correlations were observed between adiposity and servings of refined grains, and meats and alternatives such as eggs and fish, whereas negative correlations were observed with servings of vegetables, fruit, whole grains, and alternatives such as tofu, and nuts/seeds. Participants with IBD (both CD and UC) consumed more refined grains than those without IBD. Using logistic regression analysis, participants consuming more servings of vegetables and whole grains were less likely to have CD where as those consuming more serving of fruit and bean/legumes were less likely to have UC. In the Atlantic PATH cohort, which includes a region of the world with a high incidence of IBD, distinct differences in adiposity and diet quality were observed in individuals with specific types of IBD compared to those without. There is a need for collaborative efforts to address weight management and diet quality issues in those living with IBD in the Atlantic Canadian region. Both the incidence and prevalence of inflammatory bowel diseases (IBD), including Crohn’s disease (CD), ulcerative colitis (UC) and IBD-type undetermined IBDU are increasing worldwide.  There isPatients with CD often have abnormal mesenteric adipose tissue, known as fat-wrapping or "creeping fat", and mesenteric fat has been found to be a source of pro-inflammatory cytokines . PatientIn addition to excess adipose tissue, diet quality also plays a role in the regulation of the intestinal microbiome and can further augment the associated inflammatory conditions –10. DietThus, the aim of the current study was to characterize the relationship between body composition and lifestyle behaviours such as physical activity and diet in participants with CD, UC, or without IBD from Atlantic Canada, a region of the country that not only has the highest incidence in the country ,14 but hParticipants age 30 to 74 years and residents of one of the four Atlantic Canada provinces were eligible to participant in the Atlantic Partnership for Tomorrow’s Health (PATH) study. All participants provided written informed consent prior to data/sample collection. A total of 31,173 participants that were recruited into the Atlantic PATH cohort between 2009–2015 completed the baseline questionnaire which included demographic information, anthropometric data, lifestyle behaviours and medical history  as previously described [A subset of participants completed a dietary questionnaire and had physical measures  taken. To address the aim of the study, the current project involves a sub-sample of Atlantic PATH participants, who had both completed the dietary questionnaire and had body composition measured and had indicated one specific type of IBD. To allow for comparisons between CD and UC, participants that indicated having both CD and UC were excluded n = 27). Thus, the subsample included a total of 12,802 participants , showing relative enrichment of IBD within our cohort as compared with reported prevalence data exceeding 0.3% in North America 7. Thus, .Demographic  and lifestyle data  were assessed. The urban/rural classification was based on previously published work in which the Postal Code Conversion File Plus  was used to classify study participants as living in urban or rural . The lev2 were considered underweight, normal weight, overweight and obese, respectively [Anthropometric data, including height, weight, waist and hip circumference were used to calculate BMI and waist-to-hip ratio. Participants with a BMI of <18.5, 18.5–24.9, 25.0–29.9 and ≥30.0 kg/mectively . Body coDiet quality data from participants in the cohort has been previously published . In brieAll statistical analyses were performed with IBM SPSS Statistics software (version 23). Chi-square analyses were used to determine significant associations between demographic, behavioural and dietary variables in those with and without IBD. Differences in continuous anthropometric data among those with and without IBD were analyzed using ANOVA, if necessary followed by post hoc two-tailed t tests adjusted with a Bonferroni correction for multiple comparisons. Categorical variables were presented as counts (%) and continuous variables were presented as means ± standard deviation. The relationship between diet variables and adiposity was assessed by Pearson correlation coefficients. To examine the association between dietary variables and IBD, logistic regressions were used to calculate odds ratios (OR) with 95% confidence intervals. Models were unadjusted or adjusted for variables with p<0.05 or factors previously reported to impact IBD . Chronic conditions include hypertension, myocardial infarction, stroke, asthma, chronic obstructive pulmonary disease, depression, diabetes, liver cirrhosis, chronic hepatitis, irritable bowel syndrome, eczema, lupus, psoriasis, multiple sclerosis, osteoporosis, and arthritis. For the development of the statistical models for predicting the probability of having IBD, demographic variables, lifestyle behaviors, and anthropometric measures were included in the regression models. The model’s good calibration was determined by a non-significant (P>0.05) Hosmer-Lemeshow’s goodness of fit test. Only variables with a p<0.05 and that showed good model fit were retained in the final regression model. All corrected p-values <0.05 were considered statistically significant.A significant association was observed between IBD and smoking status, alcohol consumption and the number of chronic conditions. Most participants without IBD had never smoked whereas a higher percent of those with CD or UC were former smokers. A smaller percent of participants with IBD were regular/habitual alcohol consumers (33% of CD and 40% of UC) compared to nearly half (48%) of those without IBD. Of those with IBD, approximately ¼ of participants had 4 or more chronic conditions compared to less than 5% of those without IBD. There was no statistically significant association between IBD and sex, province, urban/rural, age, education, income, alcohol consumption, and physical activity level, .Several measures of adiposity were higher in participants with IBD  body mass, thus limiting its use in detecting a loss of muscle mass or strength. Furthermore, some studies have shown that in patients with IBD, BMI does not correlate well with fat-free mass  and reseOver the past decade, a clear association has been established between diet-induced obesity and the gut microbiota, and research in this area continues to grow as scientists seek to unravel the underlying mechanisms ,40. MoreThe relationship between diet and the development or management of IBD is complex and varies depending on the type and activity of the disease . A populMajor strengths of the current study include a large sample size, measured anthropometric data for assessment of adiposity, and participants from a population that is known worldwide for its prevalence of IBD. Limitations of the current study include that data are self-reported regarding disease diagnosis, physical activity level, and diet history, and its cross-sectional design. Because baseline data was used for this cross-sectional study, we currently can only identify associations, not causality. However, since participants of the Atlantic PATH cohort are part of a large longitudinal study, it will be possible in the future to examine the role of genetic, environmental, behavioural, and lifestyle factors in the development of disease .In conclusion, this is the first population-based Canadian study in adults to investigate the relationship between diet factors, body composition, other comorbidity and the prevalence of specific types of IBD in an area of the county known to have the highest incidence of the disease worldwide. Adiposity was significantly higher in participants with UC compared to those without IBD in the Atlantic PATH cohort. Findings from this study show that most participants with IBD were consuming a diet similar to those that did not have IBD, with the highest proportion of individuals meeting guidelines in the milk and dairy category. In the regression analysis, a protective effect of servings of fruit and bean/legumes was observed against UC whereas vegetables and whole grains servings were protective of CD. Ongoing and future prospective nutritional intervention studies will offer novel insights in a combined approach to optimizing IBD specific outcomes in the context of improving overall population health –52.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.


Larimichthys crocea) genes does not exist. The gene having to do with the growth efficiency of fish will have a huge impact on research. For example, the protein encoded by the IFIH1 gene is associated with the function of viral infection in the immune system, which affects the survival rate of large yellow croakers. Thus, we collected data through the published literature and combined them with a biological genetic database related to the large yellow croaker. Based on the data, we can predict new gene–trait associations which have not yet been discovered. This work will contribute to research on the growth of large yellow croakers.The importance of a gene’s impact on traits is well appreciated. Gene expression will affect the growth, immunity, reproduction and environmental resistance of some fish, and then affect the economic performance of fish-related business. Studying the connection between gene and character can help elucidate the growth of fishes. Thus far, a collected database containing large yellow croaker ( Larimichthys crocea), whose meat is delicious and has therapeutic effects, is a crucial source for the fish markets. Larger yellow croakers, belonging to Asian croakers, are economically important, however few studies focus on the genes related to economic traits of the large yellow croaker. With the development of molecular biology technology [The large yellow croaker  and the Baidu Encyclopedia (https://baike.baidu.com/). While collecting these papers, we filtered out the economic traits associated with the large yellow croaker. We used relevant genes to find data on the corresponding protein and protein sequence through Uniprot. The collected data contained the following information: reference, gene symbol/name, Uniprot Entry, protein sequence, trait, cause, pubmedID, chromosome location, and more.In order to systematically study the relationship between various traits and genes of the large yellow croaker, two contributions are made in this work. First, we collected large yellow croaker gene–trait associations that can affect economic activities related to the fish by searching the existing literature. Second, on the basis of these data, we predicted unknown gene–trait relationships. We collected papers related to the large yellow croaker through PubMed  and false positive rates  . Thus, tk value affected the prediction performance for gene–trait associations. We performed a series of comparative experiments in order to estimate the influence of k. As a result, the KATZ-YC achieved a better prediction performance when k was set as 2  when using 2-fold b and 5-fPPARa [COX2 [IL-1β [The gene–trait associations were mainly collected from published papers related to the large yellow croaker. The database contained information about the relevant genes, the corresponding traits that have been generated, and the causes for the trait  was computed in a similar way as for genes, as follows:By making an analogy between the calculating methods of gene associations and trait associations, we can use the same method to obtain connections of traits. The Gaussian interaction profile kernel similarity for traits (Pseudosciaena crocea) [KATZ measure has been successfully applied in many areas, such as link prediction of some microbial-diseases  associat crocea) . The KATKG, the trait similarity matrix KT, and the adjacent matrix A as follows:The KATZ-YC first calculated the number of walks between gene nodes  × (24 + 102) depicted the association possibilities of all the gene–trait pairs. Furthermore, we represented the partitioned matrix S′ as follows:Here, matrix S within the matrix S12, which could provide the correlative probability between each gene and trait. However, we collected a only small amount of data because the gene–trait association network is still sparse. Walks with long lengths in a sparse network may be unimportant and could perturb the link prediction. Thus, we set parameter k to be 2, 3 and 4 and evaluated the parameter k’s influence on the prediction performance. We calculated the respective unknown association probability when k was set as 2, 3 and 4 by the following formula. Here, the final prediction result matrix could be represented by matrices A, KT, and KG.We finally predicted the result for matrix Considering research about gene–trait associations of large yellow croakers was scarce, and that databases of this species were lacking, we achieved the following two tasks in this paper. First, we collected genes related to economic traits of large yellow croakers by searching various literatures and collated expressions, protein sequences, and other information associated with these genes. Second, we used a heterogeneous network to describe gene and trait similarity based on gathered known information about gene–trait associations. We further used that information to implement the KATZ-YC algorithm, improved from KATZ, in order to perform the prediction work on unknown gene–trait associations.With the development of molecular technology, more correlation between genes and traits will be found. We can build a database by collecting more data based on the abovementioned ones, and we can execute predictive models. Through the association between genes and traits, we have obtained a possible existing link between some economic traits. This work contributes to filling the void in associations between genes and economic traits of large yellow croakers. In the aquaculture of the large yellow croaker, the probability of occurrence of other traits can be predicted according to the appearance of some traits. These traits could effectively promote growth and reproduction or the prevention of possible illnesses. This understanding can further accelerate the development of the large yellow croaker breeding industry.


Here, we purified both hPSC-derived and primary mouse spinal motor neurons in parallel and used extracellular multi-electrode array (MEA) recording to compare the pharmacological sensitivity of neuronal excitability and network function. We observed similar effects for most receptor and channel agonists and antagonists, supporting the consistency between human PSC-derived and mouse primary spinal motor neuron models from a physiological perspective.Directed differentiation of human pluripotent stem cells (hPSCs) has enabled the generation of specific neuronal subtypes that approximate the intended primary mammalian cells on both the RNA and protein levels. These cells offer unique opportunities, including insights into mechanistic understanding of the early driving events in neurodegenerative disease, replacement of degenerating cell populations, and compound identification and evaluation in the context of precision medicine. However, whether the derived neurons indeed recapitulate the physiological features of the desired  The approach holds promise both for individual diseases and for subgroups as may be pursued in personalized or targeted medicine2. The potential value for such applications is particularly large in neurodegenerative diseases, for which access to early stage tissue is difficult or impossible and for which the large majority of disease cases are apparently sporadic, potentially limiting the accuracy of mouse models when mechanistic inference from rare monogenetic cases may not extend to sporadic ones. Furthermore, a growing appreciation for the effects of human-specific genes and genetic background adds additional levels of complexity that cannot be addressed in mouse models4.Deriving specific disease-relevant cellular subtypes from human pluripotent stem cells (hPSCs) may serve potentially valuable roles in disease modelling, cell replacement, and drug development5, and more recent reports have used unbiased RNA expression profiles to support the accuracy of the model motor neurons6. While the RNA transcriptome yields a valuable and unbiased view of the templates available for cellular function, differences in RNA processing, spatial restrictions, translation, and post-translational modification may yield profound differences in the integrated ensemble of cellular functions7. Indeed, the primary role of a neuron is a physiological one: to integrate synaptic inputs and generate an efferent signal transmitted to another neuron or effector cell such as muscle in the case of spinal motor neurons. How accurately stem cell models reflect the physiology of bona fide neurons remains unaddressed.Initial hPSC modeling studies identified neuronal subtypes such as spinal motor neurons based on the expression of specific protein markers8. Thus, focusing on physiology and particularly the cohort of functional ion channels in specific neurons can provide a parallel functional assessment of the value of human stem cell-based models.The rich diversity of voltage-gated ion channels, particularly potassium channels, and the specific combinations of these channels enable fine tuning of the physiological features of neuronal subpopulations to match and in some cases even define their functions9. However, a growing body of evidence supports this position in multiple neurodegenerative diseases as well, including amyotrophic lateral sclerosis (ALS)12, Parkinson’s disease13, and Alzheimer’s disease15. While a group of stem cell modelling studies have already documented abnormal motor neuron excitability in ALS, the underlying assumption that the physiology of hPSC-derived motor neurons accurately reflects the physiology of bona fide motor neurons has not been addressed. Thus, the physiological concordance of the model neurons with primary ones may be particularly important in modelling diseases of specific vulnerable neuronal types.Recent clinical phenotypes have raised the question as to whether abnormal neuronal excitability contributes to specific neurological diseases and whether the physiological features and functions of neuronal populations may contribute to their selective vulnerabilities. This possibility seems particularly strong in familial epilepsies and rare pain syndromes due to mutations in specific sodium and potassium channelsbona fide primary motor neurons and identify major determinants of excitability in each group, we compared the pharmacological profiles of purified human and mouse motor neurons using multi-electrode array (MEA) recordings. We observed a high degree of similarity between motor neurons derived from hPSCs and mice using immunocytochemistry and qPCR of ion channel transcripts. We then quantified the spontaneous activity of motor neuron cultures from each species using longitudinal MEA recording over a span of four weeks. Using a timepoint at which both mouse and human motor neuron cultures exhibited robust network activity, we first determined the contribution of multiple neurotransmitters to synchronized neuronal firing. We then identified the contributions of individual ion channel classes to intrinsic excitability and action potential firing. Although the effect sizes were sometimes larger in primary mouse motor neuron cultures than in hPSC-derived motor neuron cultures, consistent with a greater functional maturation of the primary neurons, the neurotransmitter and ion channel blockers controlling motor neuron networking and intrinsic firing were similar in both groups.To assess the similarity of hPSC-derived spinal motor neurons and 16. We leveraged this consistent selective expression to obtain highly congruent populations of mouse and human spinal motor neurons in parallel experimental paradigms :GFP mouse strain, which matches endogenous Hb9 expression18, and FACS-purified motor neurons from embryonic mouse spinal cords (12.5–13.5 days post-conception). We chose this age based on the technical feasibility to generate a sufficiently large population of motor neurons and the established use of this protocol in the literature20. FACS gating parameters were similar for both hPSC-derived and mouse primary motor neurons , which blocks the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) subset of glutamate receptors, nearly eliminated the synchronized bursts in both human and mouse motor neurons. D-(-)-2-Amino-5-phosphonopentanoic acid (D-AP5), which blocks the N-methyl-D-aspartate (NMDA) subset of glutamate receptors, yielded a partial inhibition in both species, although the effect was highly variable in mouse cultures. To assess for contamination from interneurons, we applied bicuculline, which blocks gamma-aminobutyric acid (GABA)-A receptors and did not observe a significant effect on network bursting in either species. Finally, we looked for direct electrical coupling between motor neurons using the gap junction inhibitor carbenoxolone and found that it reduced networking, consistent with the role of gap junctions in motor neuron development24. This effect was larger in mouse cultures compared with human cultures.Human and mouse motor neurons express ChAT, and acetylcholine is the primary transmitter at the neuromuscular junction 25. To address this potential explanation, instead of the HB9::GFP hPSC line, we used an Islet::tdTomato hPSC line to purify motor neurons. We found similar network properties, including a strong sensitivity to glutamatergic blockers , N-type (CaV2.2) and T-type (CaV3) calcium channels, respectively30. Effects of all three drugs were modest for both mouse and human neurons, with only the effect of nimodipine on hPSC-derived motor neurons reaching significance .KCNQ (Kv7) potassium channel openers have previously been shown to reduce motor neuron activity in both hPSC-derived and mouse motor neurons38. The HCN channel blocker ZD728839 showed a trend toward reduction of firing in hPSC motor neurons and a significant decrease in mouse motor neurons  and the WA09 (H9) Isl::tdTomato (kindly provided by J. Ichida) were maintained in mTeSR  on Matrigel  until 90% confluent, when they were either passaged or differentiated.The HUES3 HB9::GFP line3. Cells were fed daily for a period of 14 days with NB/B27: a 1:1 mixture of Neurobasal  and DMEM/F12  supplemented with GlutaMAX , NEAA , Pen/Strep , N2 , and B27 . From day 0 to day five the media was supplemented with dual SMAD inhibitors SB-431542  and LDN-193189  as well as Retinoic acid  and SAG . From day six through day 13, the dual SMAD inhibitors were replaced with DAPT  and SU-5402 . Dissociation of the 2D motor neuron culture was performed on day 14 by incubating the culture in StemPro Accutase Cell Dissociation Reagent  with DNase  for one hour at 37 °C. The resulting cell suspension was dissociated mechanically by pipetting 20 times followed by centrifugation at 200 rcf for six minutes. The cell pellet was resuspended in sterile filtered sort buffer consisting of 1% BSA , 15 mM HEPES , 1% Pen/Strep , 2 mM EDTA  in PBS  with DAPI  and filtered through a 40 µm nylon mesh  immediately prior to FACS sorting.Differentiation of hPSCs into motor neurons was performed as previously publishedFACS sorting was performed with either a BD FACSAria II or a BD FACSAria Fusion. After sorting, motor neurons were maintained in motor neuron maintenance media consisting of Neurobasal media , GlutaMAX , NEAA , N2 , B27 , and Pen/Strep  supplemented with GDNF , BDNF , and CNTF  with half media changes 3 times per week.20. After spinal cord isolation, the tissue was submerged in PBS  at 4 °C followed by incubation with Trypsin-EDTA  at 37 °C for 10 minutes with agitation. The Trypsin-EDTA mixture was then carefully replaced with 1 ml of NB/B27 with DNase  and 0.4% BSA , and the tissue was mechanically dissociated by pipetting 20 times with a fire-polished Pasteur pipette. The resulting cell suspension was filtered through a 100 µm cell strainer , centrifuged at 150 rcf for five minutes and resuspended in the same sort buffer composition as for the hPSC derived motor neurons for FACS sorting.Embryonic motor neurons were harvested from E12.5–13.5 spinal cords of B6.Cg-Tg(Hlxb9-GFP)1Tmj/J (Hb9:GFP) mice  under the dissecting microscope as previously describedThe cerebral cortex from mouse pups ranging P1-P3 was dissected and placed into HBSS  buffer containing 1% Pen/Strep  and 10 mM HEPES . The tissue was then dissociated enzymatically with 0.5 ml of 0.25% Trypsin-EDTA  for 5–8 minutes at 37 °C. The trypsin was then inactivated by adding 1.5 ml of glial media: MEM  with 10% heat inactivated donor equine serum , 3% Glucose , and 1% Pen/Strep . The resulting suspension was mechanically dissociated and then centrifuged at 200 rcf for nine mins, resuspended in glial media and slowly filtered using a 100 µm mesh filter, after which the filtrate was then seeded in tissue culture vessels coated with Poly-D-Lysine (PDL) .Cells were washed for 5 minutes in PBS and then fixed with 4% (w/v) paraformaldehyde  for 15 minutes, followed by three washes, 5 minutes each, in PbTr . Following an additional 10 minutes permeabilization with PbTr, the cells were blocked in PbTr with 5% normal donkey serum  for 45 minutes. Primary antibodies  were diluted in blocking buffer and cells were incubated overnight at 4 °C in a humidified chamber. After three 5 minute washes with PbTr, cells were incubated in the dark for an hour with secondary antibodies . Cells were finally washed three times with PbTr, and the slides were mounted in Prolong Diamond with DAPI . Slides were mounted overnight at room temperature prior to imaging.2 Profiler PCR Array Mouse Neuronal Ion Channels  and RT2 Profiler PCR Array Human Neuronal Ion Channels , respectively. RNA was extracted of sorted neurons immediately after FACS using Trizol and 2 µg of RNA were converted to cDNA with the High-Capacity cDNA Reverse Transcription Kit . qPCR was performed with iQ SYBR Green Supermix  in a Biorad CFX96 according to the array manufacturer’s protocol.Gene expression analysis of FACS sorted Hb9::GFP mouse motor neurons and HUES3 HB9::GFP hPSC-derived motor neurons was performed by qPCR array the RT48-well MEA plates (Axion Biosystems) were coated with 50 µg/ml PDL in 5 µl drops in the centers of the wells at room temperature for 1 h. After three washes with PBS, the plates were coated with laminin  for 1 hour. Sorted motor neurons  were plated in a volume of 5 µl per well and allowed to attach for 1 hour at 37 °C. Motor neuron maintenance media (see above) was added to each well to a total volume of 300 µL. Longitudinal MEA recordings were performed immediately prior to media changes three times per week by loading the MEA plate into the pre-warmed Maestro MEA plate reader (Axion Biosystems). After allowing for five minutes of equilibration, the plate was recorded for five minutes.Drugs were pre-mixed at 10x concentration in 48 well plates (see Supplemental Table For network burst analysis Fig. , we usedFor drug effects in blocked neurons Fig. , we firsSupplementary Info