Despite continued global efforts, HIV/AIDS outcomes in developing countries have not made much progress. Poor governance in recipient countries is often seen as one of the reasons for ineffectiveness of aid efforts to achieve stated objectives and desired outcomes.This study examines the impact of two important dimensions of governance – control of corruption and democratic accountability – on the effectiveness of HIV/AIDS official development assistance.An empirical analysis using dynamic panel Generalized Method of Moments estimation was conducted on 2001–2010 datasets.Control of corruption and democratic accountability revealed an independent effect and interaction with the amount of HIV/AIDS aid on incidence of HIV/AIDS, respectively, while none of the two governance variables had a significant effect on HIV/AIDS prevalence. Specifically, in countries with accountability level below −2.269, aid has a detrimental effect on incidence of HIV/AIDS.The study findings suggest that aid programs need to be preceded or at least accompanied by serious efforts to improve governance in recipient countries and that democratic accountability ought to receive more critical attention. The HIV/AIDS epidemic has been a serious threat to the world that deserves global attention. World Health Organization (WHO) statistics show that by 2013, 35 million people were living with HIV and 1.5 million people have died of AIDS  efforts have been largely ineffective; this claim is supported by academia and global development agencies , 5. ManyAs in Kaufmann's article , controlCorruption is seen as a pervasive issue, as corruptive decisions and behaviors can be committed by anyone at all levels, from high officials down to service providers at the lowest level of money flow. Even when assistance is provided by non-governmental actors bypassing recipient government, there is still room for corrupt behavior that influences aid effectiveness.Democratic accountability is yet another important governance factor in HIV/AIDS control. Because HIV/AIDS is sexually transmitted and society attaches a social stigma to it, HIV/AIDS response efforts require a multi-faceted approach that involves dedicated human resources and sustained policy commitment , 13. AlsDemocratic accountability can be realized through various means: fair election, guaranteed freedom of mass media, and active civil society . A fair Despite emphasis on relevance of governance with health outcomes including HIV/AIDS from a theoretical perspective, however, empirical evidence supporting it is lacking, and even the results of existing evidences are not consistent. For example, based on two stage least square (TSLS) analyses, Gupta et al.  showed aPrevalence and incidence of HIV/AIDS were selected for measuring HIV/AIDS aid effectiveness because they can capture the effect from all types of HIV/AIDS programs while intermediate outcomes, e.g., rate of condom usage or anti-retroviral treatment coverage, reflect effect from only particular types of programs. The unit and data source of HIV/AIDS prevalence and incidence are presented in ‘Control of corruption’ and ‘voice and accountability’ which are sub-dimensions of WGI complied by the World Bank were used as indicators for level of corruption and democratic accountability, respectively. ‘Control of corruption’ measures the perception of how much public power is exercised for private gain, including both petty and grand forms of corruption, and how much the state is captured by elites and private interests . As an iPublic and multinational HIV/AIDS disbursement per capita, which was compiled by the Institute for Health Metrics and Evaluation, was summed and converted to 2010 constant USD .The factors that previous studies had shown to be associated with HIV/AIDS outcomes theoretically and empirically were chosen as control variables. Muslims are strictly prohibited from extramarital sexual relationship and alcohol consumption and are highly likely to undergo circumcision, all of which are expected to be suppressing factors for spread of HIV/AIDS . EthniciThe level of economic development is widely recognized as one of the important factors associated with health outcomes. For example, high rates of unemployment and low wages increase the number of the population working in the sex industry and this raises the likelihood of HIV/AIDS infection . Gross nAn OLS regression was performed as an exploratory analysis although results from OLS regression are expected to be biased due to the following reasons. First, there is a possibility of endogeneity occurring by ‘simultaneity’ between some of the explanatory variables  and outcome variables . For exaWhat has been most commonly used in previous studies for addressing these biases is TSLS with instrumental variable (IV) , 30, 31.The unit of analysis is country-year. All of the explanatory variables were lagged to capture time gap in which explanatory factors take effect on the outcome . To measrt should not be allowed in GMM estimation while first-order autocorrelation can be.Generally, for an over-identified model like this, GMM is known to be more effective and hence has been used more commonly . Among tSensitivity analyses were performed to test the robustness of the results. Firstly, data were re-analyzed after outliers were removed. For this, ‘Dfbetas’ of lagged prevalence and incidence were calculated and outliers were defined as observations with Dfbetas bigger than  dataset . All theAverage HIV/AIDS prevalence of countries in the 2001–2011 dataset was 3.87%, which is quite high when compared to the global average during the same period (by approximately 0.8%). The average governance scores were lower as well . About UThe result of dynamic panel analysis differed from that of OLS analysis  and 4. UThe democratic accountability variable also showed the same pattern. While there were significant negative associations between the level of accountability and HIV/AIDS prevalence in the OLS analysis, none of the independent variables, including democratic accountability, were significant on HIV/AIDS prevalence in the GMM estimation. However, in terms of predicting the HIV/AIDS incidence, significant interaction between democratic accountability and amount of HIV/AIDS assistance was found in the GMM estimation. 1At the average level of accountability (WGI score: −0.439), more assistance money per capita can be linked to lower HIV/AIDS incidence. However, this trend turns in the opposite direction when the level of democratic accountability reaches a turning point, which is −2.120 based on the WGI accountability index. When the level of democratic accountability is 2STD subtracted from the average (WGI score: −2.269), HIV/AIDS incidence rose as the amount of assistance per capita increased. The results from the sensitivity analyses were not different as in the main analysis, except for a small change in the significance level .This paper analyzed the effect of control of corruption and democratic accountability on relationship between ODA amount and prevalence or incidence of HIV/AIDS in ODA recipient countries. Guided by economic theory, we specified a dynamic panel data model where two governance dimensions – control of corruption and democratic accountability – had significant independent or moderating effect on HIV/AIDS incidence, respectively, while on prevalence of HIV/AIDS, neither of them had a significant effect. The result may come from the ongoing emphasis on ODA funding for the HIV-AIDS preventive programs. Although a large proportion of HIV funding shifted from prevention toward the treatment and care since 1990, a prevention-focused program has still prioritized during the 2000s . Since tIn our study, the two governance dimensions showed different patterns in predicting the HIV/AIDS outcomes. While control of corruption had only independent effect on HIV/AIDS incidence, meaning that it does not have a boosting or aggravating effect on aid effectiveness, democratic accountability showed a negative interaction with amount of HIV/AIDS assistance on HIV/AIDS incidence. Such an additive effect on reducing the HIV/AIDS incidence means that aid would be more effective if it was conditioned on good democratic accountability. It is assumed that due to the nature of corruption that it is committed ‘in the dark’, improvement in the control of corruption is not easily noticed by others. A decrease in the level of corruption therefore may not have a motivational effect on other dimensions of governance. On the contrary, accountability, defined as ‘the responsibility to be able to show that they have fulfilled their original responsibility’ is a clearly visible concept . For thiFindings of our study showed that in a country with accountability level below a certain level , aid has a detrimental effect on incidence of HIV/AIDS. On the other hand, in countries with scores of above −2.269, additional aid seemed to decrease the incidence of HIV/AIDS. This is in line with the results of Kosack  and KnacFinally, our study yielded different results when we adopted OLS and dynamic panel analysis. This may mean that the presence of endogeneity and country-fixed effect in the relationship between explanatory variables and HIV/AIDS outcomes cannot be regarded as inconsequential. Finding that aid can be rather harmful on HIV/AIDS incidence in countries with low levels of democratic accountability is reaffirming the view that aid programs for control of HIV/AIDS must be preceded or at least accompanied by an effort for enhancing democratic accountability of recipient country.Findings of our study should be interpreted with consideration of the following limitations. First, because of the missing values in each variable, the number of countries that remained in the final dataset for analysis reduced significantly. Although the robustness of the results was checked through imputation of missing values, our results could not be interpreted in the same rigorous way as results that we would have from the analysis using a full dataset. Second, while our model controlled the simultaneity and country-fixed effect, there might still be remaining time-variant factors we could not identify. Therefore, focusing on the size of coefficients would be meaningless, while result of this study can be valuable in gaining an understanding of general trends and directions of variables. Third, even if our findings suggest positive effect of the two governance factors on HIV/AIDS control efforts with statistical significance in a quantifiable measure, it is also very important to look at the effect of two governance factors in each country under the individual country's specific context such as socio-economic, cultural, and political context. Qualitative studies can fill this gap and we hope that our research can be a starting point to initiate such mixed-method research in the future.In spite of the positive impact of control of corruption and democratic accountability on effectiveness of ODA as suggested in this study, it is disappointing to note that control of corruption and democratic accountability in the 56 countries has largely deteriorated since 2000 (World Bank data).Overall, it is noteworthy that future strategy for HIV/AIDS control should be planned under the careful consideration of each individual country. It is hoped that this article can be a starting point for such future research efforts.


IgG4-related disease is a novel disease entity characterized by elevated serum IgG4 and tissue infiltration by IgG4-positive plasma cells. Typical renal pathology is tubulointerstitial nephritis with storiform fibrosis, although the co-existence of various glomerular lesions has been described. Here, we present the first report of a case of IgG4-related kidney disease and membranoproliferative glomerulonephritis showing the discrepancy in IgG subclasses between the kidney interstitium and glomeruli.A 70-year-old Japanese woman was diagnosed with membranoproliferative glomerulonephritis and focal tubulointerstitial nephritis with IgG4-positive plasma cells. Immunofluorescence studies revealed predominant deposition of IgG3 and IgG2, but not IgG4 in the glomeruli. We administered oral prednisolone at 30 mg/day, and the abnormalities in urine and blood tests gradually resolved.In this case, different patterns of IgG subclasses detected in the glomeruli and interstitial plasma cells suggest overlapping immunologic abnormalities. The favorable clinical course in our patient suggests that steroid therapy is promising in cases of IgG4-related kidney disease accompanied by glomerulonephritis. IgG4-related disease (IgG4-RD) is a novel disease entity characterized by elevated serum IgG4 and tissue infiltration by IgG4-positive plasma cells. Renal involvement is often observed in this disease, and the typical histopathological findings are tubulointerstitial nephritis with abundant IgG4-positive plasma cells and storiform fibrosis and sclerosis . With reHere, we report the case of a patient diagnosed with IgG4-RD, complicated with MPGN and accompanied by focal tubulointerstitial nephritis with IgG4-positive plasma cells. We also investigated IgG subclasses in the glomeruli, and found predominant deposition of IgG3 and IgG2, but not IgG4.A 70-year-old Japanese woman with a past history of bronchial asthma and paroxysmal atrial fibrillation was transferred to Kyushu University Hospital because of leg edema and insidious onset of proteinuria and microhematuria. She had presented with mild leg edema 8 months before, and was admitted to another hospital because of pneumonia. Then heavy proteinuria was identified. She had a history of polyarthralgia, although other symptoms such as skin rash, hair loss, oral ulcers, and Sicca syndrome were not evident.4/μL, hemoglobin 8.2 g/dL, hematocrit 24.3 %, and platelets, 12.9 × 104/μL, with no atypical leukocytes. Serum biochemical analyses revealed: total protein 6.9 g/dL, serum albumin 3.0 g/dL, blood urea nitrogen 33.0 mg/dL, serum creatinine 1.09 mg/dL (eGFR 43.2 mL/min/1.73 m2), sodium 141 mmol/L, potassium 5.1 mmol/L, chloride 115 mmol/L, calcium 8.3 mg/dL, phosphate 4.9 mg/dL, and C-reactive protein 0.21 mg/dL. Quantitative immunoglobulin analysis showed: IgG 2407 mg/dL , IgA 114 mg/dL and IgM 53 mg/dL (59–269 mg/dL), and the additional measurement of IgG subclasses revealed IgG1 564 mg/dL (320–748 mg/dL), IgG2 1650 mg/dL (208–754 mg/dL), IgG3 214 mg/dL (6.6-88.3 mg/dL), and IgG4 1110 mg/dL (4.8–105 mg/dL). Serum levels of C3, C4 and total serum hemolytic activity (CH50) were 58 mg/dL (71–135 mg/dL), 9 mg/dL (11–34 mg/dL), and 32 U/mL (31.6–57.6 U/mL), respectively. Although rheumatoid factor was increased to 133 IU/mL (<15 IU/mL), antinuclear antibodies, anti-double-stranded DNA antibodies, anti-U1-ribonucleoprotein antibodies, myeloperoxidase anti-neutrophil cytoplasmic antibodies (ANCA), proteinase 3 ANCA, anti-glomerular basement membrane (GBM) antibodies, anti-Sjögren syndrome related antigen (SS)-A antibodies, anti-SS-B antibodies, circulating immune complexes, and cryoglobulins were all negative. Hepatitis B virus surface antigen, hepatitis C virus antibodies, and human T-lymphotropic virus 1 antibodies were negative. T-SPOT.TB assays for the diagnosis of latent tuberculosis was also negative. Abdominal ultrasonography and computed tomography scanning revealed the absence of systemic lymphadenopathy, and no abnormal findings in the pancreas, salivary glands, and thyroid gland, although ultrasonography showed mild enlargement of bilateral kidneys .On admission, the patient’s characteristics were: height 144 cm, body weight 38.7 kg, body temperature 36.9 °C, and blood pressure 120/46 mmHg. Physical examination revealed normal breath sounds, heart sounds, abdomen and nervous system, but signs of anemia in the palpebral conjunctiva and bilateral leg edema were found. Urinalysis revealed proteinuria (2+), and occult blood (2+). Urinary protein excretion was 2.3 g/day. Complete blood counts revealed: white blood cells 3050/μL, red blood cells 252 × 10Percutaneous kidney biopsy was performed on the 12th hospital day to determine the cause of the urinary abnormalities and kidney dysfunction. Light microscopic observation revealed global glomerulosclerosis in 1 of 13 glomeruli with mild interstitial edema and inflammation Fig. . The infBecause the patient showed extremely high serum IgG4, we performed a lip biopsy and found the diffuse infiltration of IgG4 positive plasma cell in the glandular area Fig. ; thus, t4/μL, serum albumin 3.9 g/dL, serum creatinine 0.96 mg/dL, C3 99 mg/dL, C4 23 mg/dL, IgG 1002 mg/dL and IgG4 200 mg/dL  was started. The patient showed decreased urinary protein, and increased serum albumin and complement levels. In examinations conducted in our outpatient clinic 8 months later, the patient showed proteinuria (±), hematuria (3+), white blood cell count 9690/μL, hemoglobin 11.4 g/dL, platelet count 32.5 × 10Here, we report the case of a patient diagnosed as IgG4-RD accompanied by MPGN type 1 with “full-house immunofluorescence”. We confirmed the diagnosis of IgG4-RD with serological tests and a lip biopsy showing significant infiltration of the glandular area by IgG4-positive plasma cells. IgG4-positive plasma cells with little fibrosis were also found in the kidney interstitium, and the IgG4/IgG ratio exceeded 40 %. We then diagnosed IgG4-RKD equivalent to stage A tubulointerstitial nephritis according to the schema suggested by Yamaguchi et al. . HoweverRenal involvement in IgG4-RD, designated IgG4-RKD, is characterized by increased IgG4-positive plasma cells in the kidney interstitium, and various degrees of storiform fibrosis that can be recognized with methenamine silver and trichrome stains. Several forms of concurrent glomerulonephritis have been reported in IgG4-RKD –8. MembrThe immunological abnormalities associated with IgG4-RD have been extensively investigated, and recent studies suggest an important role of T-helper (Th) 2 cells and regulatory T cells (Treg) in the development of IgG4-RD. Müller et al. demonstrated increased interleukin (IL)-4 and IL-5 levels, and upregulated IL-13 mRNA in bile samples from a patient with IgG4-related cholangitis . Tanaka With regard to the treatment, we initially started low-dose oral prednisolone 30 mg/day) which was previously reported for the treatment of IgG4-RKD 0 mg/day , after wWe have described the case of a patient with IgG4-RD accompanied by IgG4-related tubulointerstitial nephritis and MPGN with predominant IgG2 and IgG3 deposition. Different patterns of IgG subclasses detected in the glomeruli and interstitial plasma cells suggest overlapping immunologic abnormalities. IgG4-RD is a novel disease entity and the detailed pathophysiology is still unknown; thus, this emphasizes the need to accumulate the details of more cases of IgG4-RKD, and perform IgG subclass staining extensively if glomerular lesions are found in IgG4-RKDs to clarify the nature of this disease.Written informed consent was obtained from the patient for publication of this Case report and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.


We report a case of IgG4-related disease (IgG4-RD) with multiple ten-organ involvement. This case showed many clinical findings, such as bilateral swelling of salivary and lacrimal glands, autoimmune pancreatitis, interstitial nephritis, retroperitoneal fibrosis, periaortitis, systemic swelling of lymph nodes, pulmonary lesions, splenomegaly, and jejunal lesions. He was suspected as having SLE or malignant lymphoma but diagnosed as having IgG4-RD by the elevated serum IgG4 level and histological findings from kidney and lymph node. We report a case of IgG4-RD with multiple ten-organ involvement that was successfully treated with prednisolone therapy. IgG4-related disease (IgG4-RD) is characterized histologically by dense infiltration of IgG4-positive cells and fibrosis. The first report of the disease came from Japan in 2001 [A 64-year-old Japanese man complained of swelling of the submandibular glands in November 2013, and his physician decided to observe the symptoms conservatively after computed tomography (CT) revealed no abnormalities other than submandibular glands. However, swelling of submandibular glands did not improve. A blood sample in April 2014 revealed positive results for anti-nuclear antibody and pancytopenia. Connective tissue diseases such as systemic lupus erythematosus (SLE) were suspected and he was referred and admitted to our hospital in April 2014. He had never smoked, reported no allergies, and had drunk two glasses of beer daily for 30 years. He had undergone bilateral cataract surgery at 55 years old. No history of disease other than cataracts was elicited.On admission, findings were body temperature, 36.1°C; blood pressure, 109/67 mmHg; heart rate, 89 beats/min; and SpO2, 99% (room air). Physical examination showed symmetrical enlargement of the submandibular and parotid glands, which were nontender, elastic, and soft. Inguinal lymph nodes appeared swollen bilaterally. Laboratory investigations revealed pancytopenia, renal dysfunction, extremely elevated serum levels of IgG and IgG4 subclass, and hypocomplementemia . Anti-nuThese clinical, serological, and histological findings helped us to diagnose as IgG4-RD. We carried out bone marrow puncture to rule out multiple myeloma because of extremely high level of serum IgG (M protein was negative.). Plasma cells were slightly increased in bone marrow (3.8%); however, dysplasia or increased blast cells were not seen. Flow cytometry immunophenotyping of bone marrow and peripheral blood was not conducted. Multiple myeloma was able to be ruled out. We also performed biopsy of an inguinal lymph node to rule out malignant lymphoma, because of the elevated serum level of soluble IL-2 receptor. Some cells appeared slightly dyskaryotic, but we could not observe for deviation to one side or the other about light chain in the immunostaining and a proliferative quality destroying the normal structure was not seen. Malignant lymphoma was thus able to be ruled out. Numerous IgG4-positive cells were seen and IgG4/IgG ratio was over 90%. The number of IgG4-positive cells was over 300 cells/high-power field . HistoloOral prednisolone (0.6 mg/kg/day) was started as treatment for IgG-RD . SwellinIgG4-RD has been gradually recognized for the last decade. It is characterized histologically by dense infiltration of IgG4-positive cells and fibrosis, but whether infiltration of IgG4-positive cells is the reason for IgG4-RD or a response to some other inflammatory stimulations remains unclear. Few data exist on the global incidence and prevalence of IgG4-RD . Many stIgG4-RD has been described in virtually every organ system, including the salivary glands, lacrimal glands, lungs, thyroid, lymph nodes, central nervous system, pituitary body, aorta, pancreas, liver, gall bladder, bile ducts, retroperitoneum, kidney, and prostate . The preIn this case, he was suspected as having SLE because of hypocomplementemia and strongly positive result for anti-nuclear antibody, but 23.4% cases of IgG4-related disease also showed positive result for anti-nuclear antibody and about one-third of IgG4-related disease also showed hypocomplementemia . The malThe differentiation of IgG4-RD as a systemic disease from multicentric Castleman's disease or granulomatosis with polyangiitis is important. Hyper-IgG4 gammaglobulinemia and IgG4 plasma cell infiltration with fibrosis and sclerosis of tissues may be present in these diseases; thus discriminating between these diseases and IgG4-RD will be difficult in some cases. An IgG4/IgG cell ratio of >50% in the tissue, good response to glucocorticoids, and histological findings of obliterative phlebitis and a storiform pattern suggest IgG4-RD , 17.Glucocorticoids are the first line of therapy and most cases show good response, displaying improvement within a few weeks . A conseIn this case, we performed inguinal lymph node biopsy to rule out malignant lymphoma. We showed that the patient was not suffering from malignant lymphoma, but careful follow-up will still be needed because the standardized incidence ratio was 16.0 (95% CI 3.3–45.5) , suggestIn conclusion, we encountered a case with IgG4-RD that had extensive multiorgan involvements with good response to glucocorticoid. Few cases with extensive multiorgan involvements as in this case have been reported. We always have to keep in mind that this disease can happen in any organs.


From 8 to 10 June 2016, heads and representatives of states and governments, along with other key stakeholders, will assemble at the United Nations (UN) in New York, for the High-Level Meeting on Ending AIDS. There are three reasons why this meeting is an important milestone for the global response against human immunodeficiency virus (HIV) and acquired immunodeficiency syndrome (AIDS). First, the meeting will provide an opportunity to reflect on the extent of progress and unprecedented achievements that have been made in responding to the AIDS epidemic, as described in the UN Secretary-General’s report.Second, the meeting serves as an occasion to galvanize support from the global community to scale up the AIDS response. The 90–90–90 treatment target calls for 90% of people living with HIV to know their status, 90% of people who know their HIV status to have access to treatment and 90% of people on treatment to achieve suppressed viral loads by 2020.Third, the meeting will be an opportunity to reflect on specific challenges that need to be addressed as we move forward. Among them is a treatment gap and inadequate global investments in prevention.UNAIDS 2016–2021Strategy integrated efforts towards ending the AIDS epidemic fully into Transforming our world: the 2030 agenda for sustainable development. The strategy sets out the links between the HIV response and several sustainable development goals (SDGs), from SDG 1 on ending poverty to SDG 16 on promoting inclusive societies.The Building on the SDGs, we need a global approach to ending the AIDS epidemic, underpinned by universal principles of social equity and an unwavering commitment to human rights. We must pledge to invest at least 26 billion United States dollars a year in low- and middle-income countries by 2020. Diverse sources of funding, innovative forms of financing and increases in both domestic public expenditure and international assistance are all needed.Let us make history at the high-level meeting. Let us commit to charting a path forward that will secure the end of the AIDS epidemic as a public health threat in one generation.


The protocols of the crystalline sponge method, particularly those in the soaking, data collection and refinement processes, are considerably improved to give reliable structural information. e.g. temperature, time, concentration, solvents) therefore have to be optimized for every sample compound. When standard conditions do not work, a high-throughput method is useful for efficiently optimizing the soaking conditions. The X-ray experiments are also carefully re-examined. Significant improvement of the guest data quality is achieved by complete data collection at high angle regions. The appropriate disorder treatment of the most flexible ZnI2 portions of the host framework and refinement of the solvents filling the remaining void are also particularly important for obtaining better data quality. A benchmark test for the crystalline sponge method toward an achiral molecule is proposed with a guaiazulene guest, in which the guest structure (with ∼ 100% site occupancy) is refined without applying any restraints or constraints. The obtained data quality with Rint = 0.0279 and R1 = 0.0379 is comparable with that of current conventional crystallographic analysis for small molecules. Another benchmark test for this method toward a chiral molecule is also proposed with a santonin guest. The crystallographic data obtained  represents the potential ability of this method for reliable absolute structure determination.Crystalline sponges are porous metal complexes that can absorb and orient common organic molecules in their pores and make them observable by conventional X-ray structure analysis . In this study, all of the steps in the crystalline sponge method, including sponge crystal preparation, pore–solvent exchange, guest soaking, data collection and crystallographic analysis, are carefully examined and thoroughly optimized to provide reliable and meaningful chemical information as chemical crystallography. Major improvements in the method have been made in the guest-soaking and data-collection steps. In the soaking step, obtaining a high site occupancy of the guest is particularly important, and dominant parameters for guest soaking ( In 2013, we reported a new technique for single-crystal X-ray diffraction analysis that does not require the crystallization of samples in the sample preparation  host crystal synthesis, (2) solvent exchange and (3) inclusion of the target molecules to be analyzed. Although the detailed procedures in each step have been described in a previous paper (Inokuma 2.1.1.2)3(tpt)2·x(solvent)]n (1) 2·x(solvent)]n (2)  complex 1 in most cases as the crystalline sponge. In addition to the relatively low symmetry of the space group, complex 1 is a suitable crystalline sponge because of the following advantages in host–guest complexation in the pores: (1) The size of the pores  is appropriate for the accommodation of organic molecules of common sizes. (2) The tpt ligand offers flat and electron-deficient binding sites suitable for stacking with aromatic compounds and for CH–π interactions even with aliphatic compounds  The framework is relatively flexible and guests are often bound in the cavity by induced-fit molecular recognition. Regarding the pore size (5 × 8 Å), it does not strictly limit the size of guest to be included because the sponge framework is very flexible and guest molecules larger than the portal are often accommodated by expanding the pore size. Typical expansion of the pores by inclusion of guaiazulene and santonin is visualized in Fig. 2In our original paper, two porous complexes, [ solution of the tpt ligand at room temperature in a test tube 3(tpt)2·x(solvent)]n 3(tpt)2·x(solvent)]n  smooth and complete solvent exchange in the pores, (2) high guest occupancy in the pores, (3) thorough thermodynamic equilibration of the absorbed guest, and (4) careful data collection particularly at high-angle regions. It is worth noting that tailored soaking conditions for every target guest should be designed based on the understanding of host–guest chemistry as well as on feedback from X-ray crystallographic experiments. As such, unlike for common X-ray crystal structure analysis, sophistication and craftsmanship are required not only in crystallography but also in host–guest and synthetic chemistry for successful analysis. The technique should be ideally performed (at least directed) by scientists who can survey these different fields. We recommend the guest soaking and crystal structure analysis be performed by the same researcher.2.1.3.supporting information shows that the signal at 1346 cm–1, assignable to nitrobenzene, almost completely disappears after soaking in cyclohexane for 1 week at 50°C. The complete solvent exchange can be confirmed by elemental analysis or even by crystallographic analysis. Single-crystal X-ray analysis confirms the presence of ordered cyclohexane molecules in the pores, which indicates successful solvent exchange . After completing the solvent exchange with cyclohexane, the crystalline sponge is ready to use. Clardy reported that the solvent exchange step can be omitted if the crystalline sponge is synthesized in chloroform–methanol . The solvent exchanging process can be monitored by IR spectroscopy; Fig. S1 of the et al., 2006The yield of high quality crystals suitable for the crystalline sponge method is actually not very high (< 5%) and, as in common single-crystal X-ray experiments, researchers often have to carefully choose the top quality crystals from many crystals under a microscope. It is rather an advantage of the crystalline sponge method that this quite inefficient step  can be undertaken before guest absorption. Namely, the required amount of the sample can be reduced to nano-to-microgram quantities by undertaking the crystal selection first and the guest soaking second. In this aspect, the crystalline sponge method is clearly distinguished from cocrystallization methods  Original method: The prototype of the crystalline sponge method can be ascribed to the observation of single-crystal-to-single-crystal (SCSC) solvent exchange in the pores of porous complex (1), which we reported in 2002  show remarkable guest binding properties in aqueous solution . However, this original method is still useful and can be the method of choice when a large quantity of the target compound is available. There is no technical difficulty in this original method.(ii) A-grain-of-crystal method: To establish the method for the structural analysis of small molecules, the required amount of the sample should be reduced. This is especially necessary when the target compounds are highly valuable and obtained only in minute quantities; this is the case when analyzing isolated natural products, metabolites and impurities in drugs and foods, or flavor components. Thus, we reduced the experimental scale of the original method to the extreme by performing the guest-soaking experiment with only one grain of the sponge crystal . From the crude solvent-exchanged sponge crystals, a top quality crystal was chosen and contacted with a small amount  of neat liquid sample or a supersaturated viscous solution on a glass plate. The guest absorption is usually completed in minutes to hours, although depending on the samples and crystal size it can sometimes take a few days. It is noteworthy that the order of events in the original method  is reversed in the ‘a-grain-of-crystal’ method , and this reversed protocol made it possible to analyze very small amounts of the target compounds. The method itself is technically not particularly difficult because there are only a limited number of parameters that influence the data quality. Slow evaporation method: In the above method, the quality of the crystals often deteriorates severely during guest absorption if the host–guest interaction is too strong and the host framework is mechanically stressed. To perform the guest absorption with [(ZnI2)3(tpt)2·x(solvent)]n (1) under much milder conditions, the crystals can be soaked in a diluted solution of the guest . The use of a poor solvent is essential because otherwise the guest is extracted from the crystal; cyclohexane with a minimum amount of CH2Cl2 is typically used as the solvent. In this method, the sponge crystal is placed in a vial and soaked in the solution of the guest. The vial is equipped with a needle, through which the solvent slowly evaporates. During this slow evaporation, the guest is gradually concentrated, finally reaching the saturation point, and is forced to diffuse into the crystal. The data quality in the crystalline sponge method depends highly on this guest-soaking step. As emphasized in the general discussion of the standard protocols, there is no all-purpose protocol for this soaking step that can be applied to all compounds. The guest-soaking time ranges from minutes to days, or sometimes even weeks.Typically, a set of several conditions are examined. The parameters include soaking temperature, soaking time, solvent, concentration, crystal size, evaporation rate (controllable by changing the thickness of the needle). Guest absorption is, in general, faster at higher temperatures but the guest seems to be more concentrated and ordered in the crystal after soaking at lower temperatures. Annealing of the crystals during the guest-soaking is sometimes effective . The guest concentration in the crystal can be roughly estimated by microscopic IR measurement and this helps in the optimization of the soaking conditions. Although this slow evaporation method has considerably expanded the scope of the crystalline sponge method, it is technically difficult because every step in the method is unfamiliar and the optimization of the soaking conditions requires experience. We recommend that those new to the crystalline sponge method experience the guest-soaking techniques in the order described here  and [101] directions, respectively, in the sponge crystal . The faces indexed as (010) and (101) are not surfaces of a spontaneously grown crystal of 1. However, (010) is considered as a cleavage face because a surface indexed as (010) is found in many split crystals. A typical crystal of 1 with face indexes is shown in Fig. 5Two channels  face of the crystal. Several different sites and modes of guest binding are observed in the pores of the crystalline sponge . The pyridyl protons on the tpt ligand are good hydrogen-bond donors and are often located within a hydrogen-bond distance from the nitrogen and oxygen lone pairs of the substrates . Even the Br atom of the guest can be a hydrogen-bond acceptor . The iodine atom of the ZnI2 center seems to be a good hydrogen-bond acceptor . The host network is relatively flexible and is quite often deformed before and after the guest absorption, which results in efficient induced-fit molecular recognition .As rod-like molecules often lie along the channel ge Fig. 6. The mos65 Fig. 6a. The pes Fig. 6b. Even or Fig. 6c. The ior Fig. 6d. The hn Figs. 6e and f.via reversible host–guest complexation. The binding sites in the pores vary case-by-case and are unpredictable for any guest. When a large association constant is gained at the binding site, the guest is trapped and concentrated. In other words, finding at least one suitable strong binding site is essential for every guest to be ordered and crystallographically analyzed.When the sponge crystal is soaked in a guest solution, the guest molecules can freely diffuse into the solvent-filled pores and find the most suitable binding sites 2.2.3.Although the details of the structural analysis of the guest-soaked crystalline sponge vary case-by-case depending on the guest examined, common and general methods for the analysis can be suggested. Taking guaiazulene as an example, a general procedure for the analysis is described. We recommend that the data for guaiazulene be used as a benchmark for evaluating guest soaking and crystal structure analysis in the crystalline sponge method. Another benchmark test for this method toward a chiral molecule is also proposed with a santonin guest. The obtained crystallographic data represents the potential ability of this method for reliable absolute structure determination.2.2.4.1 is labile and rapidly deteriorates when it is taken out of the cyclohexane solvent because the solvent is lost from the pores by rapid evaporation. Upon inclusion of non-volatile guests, however, the crystals become relatively stable and can be treated with various protectants. After coating with an appropriate protectant, the guest-absorbed crystal is subjected to data collection. For the guaiazulene-containing crystals, we used Hampton Type A low viscosity oil as a protectant.Cyclohexane-filled crystalline sponge I/σ value at the high-angle region (d < 1.0) is particularly important to obtain high data quality for the trapped guest in the crystalline sponge as reflections from a guest is much weaker than those from the host at high-angle region. Although Mo Kα radiation is commonly used for the X-ray structure analysis of metal complexes because of the weaker X-ray absorption by heavy atoms, Cu Kα radiation has provided, overall, better results in the crystalline sponge method. The crystalline sponge is a weakly diffracting crystal because of the huge number of solvent molecules  in the pores. Thus, the value of I/σ at the high-angle region by the Mo Kα radiation is typically 1 or lower. However, Cu Kα radiation provides a much higher I/σ value for the high-angle region. For guaiazulene, an exposure time of 90 s per data frame was applied for the high-angle region to satisfy the condition I/σ > 10. Under these conditions, high-resolution data were collected for guaiazulene with sufficient intensity, and the 0.82–0.79 Å shell exhibited a completeness of ∼ 90%. More than 1 d (27 h) was required for complete collection of the above data.Several different X-ray source/detector combinations have been examined for the X-ray diffraction data collection. Data collection with a sufficient number of reflections and a suitable et al., 2015abetc.) mean that it may be less effective than a good in-house facility. Prompt feedbacks from the crystal structure analysis to the soaking experiments are very important to optimize the soaking conditions. Also, beam-times for experiments at a synchrotron facility are usually limited, and highly experienced operations are necessary to find the suitable conditions for data collection . All the data in Table 1Kα radiation source.Using a synchrotron X-ray source  but geometrical distortion in the framework is not obvious. This slight change of framework causes a pseudo-symmetry issue (faint C2/c symmetry) and may lead to misassignment of the space group. The space group of the guest-included crystalline sponge should be carefully determined based on the extinction rule without excessively relying on an automatic suggestion by a space-group determination program.The crystalline sponge has the centrosymmetric 2.2.6.SHELXT, . After this disorder treatment on the ZnI2 fragments, a partial or full guest molecule clearly appears in the difference electron density map. In the analysis of the guaiazulene-absorbed crystalline sponge, the initial structure was obtained as described above.After obtaining an initial structure for refinement using a standard program  were analyzed with good data qualities  nevertheless gave an acceptable fit to the model, which was constructed based on the chemical knowledge that the compound had a flavone (4H-chromen-4-one) skeleton. Although crystallographically poor, structural information gained from the electron densities is of great help for natural product chemists, particularly when the structural information is supported by or combined with other measurements such as NMR and MS. In the present study, pure chemical compounds 5–7 purchased from a reagent company were used.(s Table 1. In the ii) Cubane: The structure analysis of cubane (11) is worthy of comment. Despite the high guest occupancies in the four sites  and the appropriate treatment of void-filling solvents, the structure analysis resulted in a seemingly worse R1 value (0.1296). The reason for this moderate R1 value is due to the significant one-dimensional streak-shaped residual electron densities around ZnI2 portions in the difference electron density map. These residual densities suggest severe one-dimensional disorder of these parts. In the reciprocal space, diffused scattering along the pore directions  are found. These results suggest one-dimensional periodicity loss involving geometrical uncertainty of ZnI2 portions.(R1 reached 0.0530 and 0.0546 for each phase). Reconstructed precession images for 1 and 11  represented that no twinning was found in 1. Guest-induced twinning might be considerable in the crystalline sponge method.By applying data treatment as a twinning crystal, crystallographic data was improved (iii) Others: In our original report, one of the authors attempted the structure analysis of six compounds (8–13) using the sponge method without any additional chemical information. Although scientifically not particularly important, this attempt at ab initio structure analysis did allow us to learn some limitations and warnings for the method. In the analysis of 8, for example, we were unable to distinguish whether the compound was a phenol, an aniline or a toluene. By combination with MS data, however, an aniline structure was correctly elucidated. In the updated data, the observed Csp2—Nsp3 bond length is 1.33 (3) Å, and is clearly discriminated from the Csp2—Csp3 bonds in the structure [1.57 (5) and 1.52 (4) Å]. would be helpful for determining the three-dimensional structure of target compounds in general crystallographic structure analysis. Expected advantages of this method are (i) determination of the absolute structure of a molecule consisting of only light atoms and (ii) direct observation of the molecular structure of an oily compound. The crystalline sponge method would effectively work for those purposes. We also note that the protocols discussed here are still tentative and should be elaborated through future discussions among users and by many successful results.The full optimization of all the steps for every compound is however not always easy. For practical reasons, also depending on the purpose, researchers may have to treat unoptimized data. In such cases, researchers should not forget the important major premise that et al., 2015The crystallographic analysis of ‘post-crystallized’ compounds in the pores with low site-occupancies is quite an unusual situation, and one that past crystallographers have seldom experienced. Researchers may encounter crystallographic phenomena or problems, including space-group changes, the formation of superlattice structure, pseudo-symmetry problems, treatment of guests lying on symmetry elements, and treatment of guests with low site occupancies, all of which were raised upon guest inclusion into the sponge crystal and may be unfamiliar and unexpected to chemists. In addition, the treatment of solvents that fill the void space is a common yet tedious problem in MOF structure analysis  cyclohexane, guaiazulene, santonin, nobiletin, tangeretin, heptamethoxyflavone, diisopropylaniline, nitrobenzaldehyde, cinnamaldehyde, Dimethyl_cubanedicarboxylate, cubane_twin_1, cubane_twin_2, bromophenanthrene, vanillin. DOI: 10.1107/S2052252515024379/de5035Dimethyl_cubanedicarboxylatesup2.hklStructure factors: contains datablock(s) Dimethyl_cubanedicarboxylate. DOI: 10.1107/S2052252515024379/de5035cyclohexanesup3.hklStructure factors: contains datablock(s) cyclohexane. DOI: 10.1107/S2052252515024379/de5035guaiazulenesup4.hklStructure factors: contains datablock(s) guaiazulene. DOI: 10.1107/S2052252515024379/de5035santoninsup5.hklStructure factors: contains datablock(s) santonin. DOI: 10.1107/S2052252515024379/de5035nobiletinsup6.hklStructure factors: contains datablock(s) nobiletin. DOI: 10.1107/S2052252515024379/de5035tangeretinsup7.hklStructure factors: contains datablock(s) tangeretin. DOI: 10.1107/S2052252515024379/de5035heptamethoxyflavonesup8.hklStructure factors: contains datablock(s) heptamethoxyflavone. DOI: 10.1107/S2052252515024379/de5035diisopropylanilinesup9.hklStructure factors: contains datablock(s) diisopropylaniline. DOI: 10.1107/S2052252515024379/de5035nitrobenzaldehydesup10.hklStructure factors: contains datablock(s) nitrobenzaldehyde. DOI: 10.1107/S2052252515024379/de5035cinnamaldehydesup11.hklStructure factors: contains datablock(s) cinnamaldehyde. DOI: 10.1107/S2052252515024379/de5035cubane_twin_1sup12.hklStructure factors: contains datablock(s) cubane_twin_1. DOI: 10.1107/S2052252515024379/de5035cubane_twin_2sup13.hklStructure factors: contains datablock(s) cubane_twin_2. DOI: 10.1107/S2052252515024379/de5035bromophenanthrenesup14.hklStructure factors: contains datablock(s) bromophenanthrene. DOI: 10.1107/S2052252515024379/de5035vanillinsup15.hklStructure factors: contains datablock(s) vanillin. DOI: 1418972, 1418974, 1418979, 1418978, 1418980, 1418976, 1418973, 1418977, 1418970, 1418971, 1442316, 1442317, 1418969, 1418981CCDC references:


APOE, BDNF, CHRNA4, COMT, DRD4, HTR4, IGF2, MAOA, SLC5A7, SLC6A3, and SNAP25. We find significant associations with variability with markers on nine and we discuss the results in terms of neurotransmitters associated with each gene and the characteristics of the associated measures from the reflexive attention task.Individuals are not perfectly consistent, and interindividual variability is a common feature in all varieties of human behavior. Some individuals respond more variably than others, however, and this difference may be important to understanding how the brain works. In this paper, we explore genetic contributions to response time (RT) slope variability on a reflexive attention task. We are interested in such variability because we believe it is an important part of the overall picture of attention that, if understood, has the potential to improve intervention for those with attentional deficits. Genetic association studies are valuable in discovering biological pathways of variability and several studies have found such associations with a sustained attention task. Here, we expand our knowledge to include a reflexive attention task. We ask whether specific candidate genes are associated with interindividual variability on a childhood reflexive attention task in 9–16 year olds. The genetic makers considered are on 11 genes:  Interindividual variability occurs in a variety of human behaviors, including how we pay attention. Such variability may arise because it takes effort to maintain focus while attending to certain tasks, and there are periodic lapses in the ability to sustain that effort . VariabiThe variability captured during a reflexive attention task is an important component of attention . It can be measured using a modification of Posner’s orienting task . In thesslope variability, which refers to the change in a participant’s RT over the course of the task. It might be observed, for example, that early task RTs are faster than later task RTs  will include neurotransmitters whose availability make neural transmission more or less efficient. Two types of genetic markers are single nucleotide polymorphisms (SNPs) and variable number tandem repeats (VNTRs). A SNP is a variation at a single point  in the genetic sequence. A VNTR is a variation in the number of copies of a short string of nucleotides. The nucleotide (for SNPs) and number of copies of a repeat (for VNTRs) is inherited from parents.COMT (which catabolizes dopamine) is associated with increased RT variability on the CPT in the general population. This is significant because it indicates that research that may lead to pharmacological treatments can begin by studying very specific deficits (endophenotypes) , and SNAP25). These genes are related either to the availability of neurotransmitters such as acetylcholine, dopamine, and serotonin and to growth, brain development, attention, or general cognition. Multiple markers were used for each gene. These markers are SNPs and VNTRs. Risk alleles for some aspect of attention or cognition are known for 23 of the 39 markers.The markers under consideration were on 11 genes . Each kit collects approximately 2 ml of saliva, which can then be purified to obtain DNA. Nucleotides can be tagged with a fluorescent dye. Genotyping was performed using the GoldenGate assay on the BeadXpress system . Once the array had been visualized with the BeadXpress reader, wavelength and intensity values of the fluorescence were used to determine genotype. Allele detection and genotype calling were performed using GenomeStudio software v2011.1 . Genotype was coded as an ordinal variable with zero indicating no genetic risk, one indicating intermediate risk, and two indicating higher risk of attentional deficits.As described under Recruitment, we used a modified extreme group design including a limited number of participants from the middle of the infant PC distribution. This is an oversampling of the extreme ends of the distribution. Those in the extreme groups might need intervention in the sense that (if we could determine infant indicators of child difficulties) we could try to prevent infants from developing problems as children. However, we did not specifically try to recruit children who were having such problems because the traits we are interested in are likely normally distributed in the population. Using a specific endophenotype rather than a diagnosis to define groups is a genetically sound practice.Statistically, by minimizing the within-group variance and maximizing the between group differences in the predictor variables it becomes easier to look at the relations between predictors and outcomes. Even though some researchers have discouraged the use of extreme group designs, the use of a continuous variable and an oversampling of the extremes is statistically justified . We havePrior to analyses, RTs were prepared as follows. Only RTs of correct trials were analyzed for genetic association. Correct responses were those for which the participant (1) selected the side on which the target appeared and (2) responded between 200 and 1000 msec after the target appeared. It is nearly impossible for a child to respond to a target in under 200 msec and so RTs lower than this were assumed to be anticipatory. Likewise, RTs longer than 1000 msec are quite slow for this type of task and were assumed to represent lapses in attention.To examine slope we used a mixed (multilevel) model analysis. A multilevel model analysis is similar to a regression analysis but accounts for the nested nature of the data. We used this method because an intraclass correlation indicated that 31% of the variance came from the individual (level 2) data and we needed to take this into account when analyzing trial level (level 1) data. We entered all 39 markers, sleepiness, age, trial number, infant percent correct score, and the interaction between trial number and genetic marker as predictors of RT. However, because we were interested in slopes over the course of the task, we only examined output pertaining to the interaction between the genetic marker and trial number. Models for each of the seven outcome variables were tested separately using SPSS version 21.p-values greater than .36. This indicates that, given the minor allele frequency based on a Caucasian population, the observed genotypes are not significantly different from the expected genotypes.In this study, all SNPs were in Hardy-Weinberg equilibrium with all M = 7.83%, SD = 6.64%), therefore, based on the distribution of error rates we excluded data from children with an error rate over 40% (n = 5). Of the children whose data were included, 50.92% were male. Their ages ranged from 9 to 16 years .Average error rate per child was highly variable . Because RTs were somewhat skewed , the logarithm of RT was used for statistical analyses so that the data met assumptions for normality. As expected, skewness was reduced, skewness = 0.04, SE = 0.18.Using only included participants, RTs ranged from 200 to 999 milliseconds , and all models passed Bonferroni correction. Because our primary question involved genetic associations with RT slope, we examined the interaction between genotype and trial number in predicting log transformed RT for each of seven outcome measures. Five of seven mixed multilevel models contained a total of 21 significant associations . We entered 39 genetic markers, sleepiness, age, infant percent correct score, trial number and the interaction between trial number and genetic marker as predictors of log transformed RT. The improvement of each model over a baseline model with no predictors was significant  was significantly associated with markers on nine genes. As can be seen in APOE, both involving the rs7412 SNP. Rs7412 genotype groups differed significantly for slopes on the Single Bright Invalid condition, F  = 3.58, p = .03. Mean slope RT for those with zero risk alleles was 0.62 msec (SD = 0.02 msec), although there were only two individuals in this genotype group. Nevertheless, slope for those with one risk allele is in a different direction  from those with two risk alleles . Only the two risk allele group is improving over the course of the task .There were two associations with markers on APOE rs7412 genotype groups also differed significantly for slopes corresponding to the Single Dim Invalid condition, F  = 3.28, p = .04. Slope RT for those with zero risk alleles was 0.65 msec (SD = 0.19 msec). Slope RT for those with one risk allele was 0.19 msec (SD = 0.48 msec), and was 0.01 msec (SD = 0.40 msec) for those with two risk alleles. No individuals were improving over the course of the task for this condition, but the two-risk allele group showed the smallest increase in RT.The CHRNA4 gene likewise had a single marker (rs6090387) which was associated with two outcomes. For Single Dim Invalid , those with no risk allele had a slope of 0.01 msec (SD = 0.40 msec), while those with two risk alleles had a slope of 0.23 msec (SD = 0.31 msec) and heterozygotes had slopes between these two extremes . Likewise, Single Dim Valid  had a significant linear trend in the same direction. Those with no risk alleles had a slope of -0.24 msec (SD = 0.41 msec), those with two risk alleles had slopes of -0.03 msec (SD = 0.36 msec), and heterozygotes had slopes between these two extremes, M = -0.17 msec; SD = 0.44 msec. This is a linear trend showing worse performance the more copies of the risk allele.The SLC5A7 gene had two SNPs significant for differential association with slopes by genotype. Rs3806536 was associated with Neutral Bright, F  = 3.50, p = .03. The risk allele for this SNP is unknown for attentional deficits. Slope RT for those with the AA genotype was -0.04 msec (SD = 0.32 msec). Slope for those with the AG genotype was -0.14 msec (SD = 0.41 msec) and -0.16 msec (SD = 0.33 msec) for those with the GG genotype. In other words, those with the AA genotype showed the least improvement over the course of the task. Rs4676169 was associated with the No Cue condition, F  = 4.69, p = .01 and with the Neutral Bright condition, F  = 3.70, p = .03. For the No Cue condition, those with the AA genotype has slopes of 0.04 msec (SD = 0.42 msec). Those with the AG genotype had slopes of -0.15 msec (SD = 0.47 msec) and those with the GG genotype had slopes of .03 msec (SD = .70 msec). For the Neutral Bright condition those with the AA genotype had slopes of 0.02 msec (SD = 0.40 msec). Those with the AG genotype had slopes of -0.15 msec (SD = 0.37 msec) and those with the GG genotype had slopes of -0.10 msec (SD = 0.33 msec). In both cases the heterozygotes improved in RT across the task. This pattern of results is termed heterozygous advantage.The SNAP25 gene is associated with all three neurotransmitters discussed in this paper. It had one SNP significant for differential association with two outcomes. Rs6077690 was significantly associated with slopes for Single Dim Invalid  and Single Dim Valid, F  = 3.73, p = .02. For the association with Single Dim Invalid, the slope for those with the zero risk alleles the slope was 0.06 msec (SD = 0.43 msec). For those with one risk allele the slope was 0.03 msec (SD = 0.41 msec), and for those with two risk alleles the slopes was 0.00 msec (SD = 0.40 msec). For Single Dim Invalid, those with two risk alleles are not slowing over the course of the task while the other two genotype groups are. This is an overall linear trend. For Single Dim Valid, the linear trend is reversed so that those with two risk alleles are making the least improvement over the task. Slope for the zero risk allele group is -0.20 msec (SD = 0.36 msec). For the one risk allele group the slope is -0.22 msec (SD = 0.47 msec) and for the two risk allele group the slope is -0.11 msec (SD = 0.33 msec). This pattern of results is consistent with the risk allele as established in the literature.The DRD4 rs1800955 is significantly associated with slopes for the No Cue condition  and for the Neutral Bright condition, F  = 3.27, p = .04. For the No Cue condition, slope RT for those with zero risk alleles was -0.01 msec (SD = 0.38 msec). Slope RT for those with one risk allele was -0.17 msec (SD = 0.45 msec), and was 0.08 msec (SD = 0.80 msec) for those with two risk alleles. Those with two risk alleles get slower over the course of the task while heterozygotes made the most improvement. While this is consistent with the risk allele as identified in the literature, the pattern of results also shows heterozygous advantage. For Neutral Bright, those with zero risk alleles had slopes of -0.11 msec (SD = 0.32 msec); those with one risk allele had slopes of -0.15 msec (SD = 0.38 msec); and those with two risk alleles had slopes of -0.03 msec (SD = 0.41 msec). This also shows a pattern consistent with heterozygous advantage.The IGF2 gene had one SNP significant for differential association with slopes by genotype. Rs734351 was associated with No Cue, F  = 3.39, p = .03. The risk allele for this SNP has not been established. Slope RT for those with the CC genotype was -0.05 msec (SD = 0.85 msec). Slope for heterozygotes was -0.01 msec (SD = 0.39 msec), and -0.14 (SD = 0.52 msec) for those with the TT genotype. This indicates that those with the TT genotype are getting the fastest over the course of the task.The SLC6A3 had six significant associations on four outcome measures. The SLC6A3 VNTR was significantly associated with slopes for the No Cue condition, F  = 3.00, p = .049. Those with no risk alleles  had slopes of -0.24 msec (SD = 0.60 msec). Heterozygotes had slopes of -0.04 msec (SD = 0.67 msec) and those with two risk alleles had slopes of -0.06 msec (SD = 0.45 msec). That is, those with two risk alleles are not getting faster over the course of the task for this condition as much as those with no risk alleles.F  = 4.44, p = .01. The risk allele for this SNP has not been established; however, those with the CC genotype did not improve on RTs over the course of the task  and those with the TT genotype slowed down over the course of the task . Heterozygotes, however, did improve . This pattern is heterozygous advantage.For rs2042449 there were also significant differences by genotype on slopes for the Single Bright Invalid condition, SLC6A3 showed significant differences by genotype for Single Bright Invalid  and Neutral Bright, F  = 4.50, p = .01. Slopes for Single Bright Invalid were -0.01 msec (SD = 0.45 msec) for the zero risk allele group. Slopes for the two risk allele group were -0.03 msec (SD = 0.41 msec). Heterozygotes had slopes of 0.30 msec (SD = 0.52 msec), but there were only two participants in this group. This pattern is suggestive of a heterozygote disadvantage, but the heterozygote group is so small that it is difficult to say for sure. For Neutral Bright, those in the zero risk group had slopes of -0.07 msec (SD = 0.41 msec); heterozygotes had slopes of -0.03 msec (SD = 0.21 msec); and those in the two risk group had slopes of -0.13 msec (SD = 0.34 msec). For this condition results are opposite those expected for sustained attention.Rs2617605 on SLC6A3 was significant for slopes on Neutral Bright . Slopes for those with zero risk alleles were 0.02 msec (SD = 0.37 msec) while those with one risk allele had slopes of -0.13 msec (SD = 0.38 msec) and those with two risk alleles had slopes of -0.14 msec (SD = 0.35 msec). This is the opposite direction of effect from established risk.Rs2937639 on SLC6A3 was significant for slopes on Single Dim Valid, F  = 3.69, p = .03. Slopes for those with zero risk alleles were 0.27 msec (but there was only one participant in this genotype group). Nevertheless, those with one risk allele had slopes of -0.18 msec (SD = 0.34 msec) and those with two risk alleles had slopes of -0.21 msec (SD = 0.43 msec). Both genotype groups are getting faster, but the two risk allele group improved more.Rs6350 on BDNF gene had two SNPs significant for differential association with slopes by genotype. Rs2203877 was associated with slopes for the Neutral Bright condition  = 3.13, p = .04) and rs6265 was also associated with slopes for the Neutral Bright condition, F  = 3.61, p = .03. For rs2203877, the risk allele is unknown. Slope RT for those with the CC genotype was -0.11 msec (SD = 0.37 msec). Slope for heterozygotes was -0.10 msec (SD = 0.41 msec), and slope for those with the TT genotype was -0.13 msec (SD = 0.30 msec). In this pattern, C appears to be the risk allele for this condition with a reflexive attention task. For rs6265 the zero risk allele group showed no improvement in RT over the course of the task . For the one risk allele group slope was -0.12 msec (SD = 0.34 msec). For the two risk allele group, slope was -0.11 msec (SD = .39 msec). This pattern is opposite the expected based on the risk allele.The HTR4 rs1862345 is significantly associated with slopes for the Single Dim Valid condition, F  = 4.17, p = .02. The risk allele is unknown. Those with the AA genotype had a slope of -0.18 msec (SD = 0.44). Those with the AT genotype had a slope of -0.24 msec (SD = 0.39 msec). Those with the TT genotype had a slope of -0.13 msec (SD = 0.43 msec). This pattern of results suggests heterozygous advantage.COMT, DRD4, IGF2, MAOA, SLC6A3, and SNAP25), which have been established as associated with some aspects of attention [BDNF, HTR4, MAOA and SNAP25 have been implicated in serotonin pathways, which have also been associated with attention [APOE, SLC5A7, CHRNA4, and SNAP25 have been connected to acetylcholine pathways, likewise associated with attention [Here we have shown that genetic markers influence slope variability on a reflexive attention task. Our findings are unlikely to be due solely to chance because (1) the models reported survived correction for multiple comparisons, (2) entering all genetic markers in a single model negates the need for correction for multiple comparisons within that model, and (3) they are biologically plausible. Regarding this third point, we tested genes involved in dopamine pathways  or for similar effects on the SLC5A7 associated with acetylcholine and the HTR4 gene associated with serotonin. This idea is explained later in more detail but essentially involves the idea that either too little or too much dopamine is problematic. Other findings might be explained by differences between this task and the task with which the gene  was previously associated in the literature . Risk was established with attentional components in disorders such as Alzheimer’s disease [There were five of seven task outcomes that had three or more significantly associated genetic markers: Neutral Bright, No Cue, Single Bright Invalid, Single Dim Invalid, and Single Dim Valid. In the following paragraphs, we explore these findings including our observation that some of the significant findings can be explained by optimal dopamine levels  and slope on five outcome measures: Neutral Bright (7 associations), No Cue (4 associations), Single Bright Invalid (3 associations), Single Dim Invalid (3 associations), and Single Dim Valid (4 associations).The Neutral Bright condition involves two equiluminant cues, one on each side of the display. Which side the target appears on is at chance. Participants might get slower over the course of the task for this condition more than for neutral dim if the bright cues become harder and harder to ignore over the course of the task. This might occur, for example, if the bright cues induce fatigue. Related ideas have been explored in studies dealing with visual fatigue associated with computer use –52, but The No Cue condition entails the target appearing after a variable delay from the previous trial. This condition might be especially difficult for participants who rely on a temporal warning that the target is about to appear, although  it also does not provide reliable location information.The Single Bright Invalid condition presents a cue contralateral to where the target subsequently appears. It provides a temporal cue but the spatial information is misleading. Some participants may get worse at this condition over the course of the task if fatigue makes it harder and harder to ignore invalid cues. The Single Dim Invalid condition also involves a cue appearing contralateral to where the target will subsequently appear but uses a dim cue. We anticipated that dim cues (which are still clearly visible) might be more sensitive at distinguishing between genotypes.The Single Dim Valid condition involves an ipsilateral cue preceding a target. Typically in this situation, participants are faster to respond than in a Single Dim Invalid or No Cue condition because attention is already in the correct location for responding to the target—there is no need to ignore the cue and attend only to the target. However, fatigue could make a participant respond more slowly over the course of the task. Once again, we will discuss the results by neurotransmitter.APOE, CHRNA4, SLC5A7, and SNAP25), two were significantly associated with Single Dim Invalid, and one each with No Cue, Neutral Bright, Single Bright Invalid, and Single Dim Valid. In the case of SLC5A7, two different gens on the same gene were associated with the same outcome measure .Of the three genes associated with acetylcholine, (APOE gene and slope outcomes. This gene is associated with the neurotransmitter acetylcholine and has been associated both with Alzheimer’s disease (which has attention-related symptoms) and with attention in healthy adults prior to any signs of Alzheimer’s disease [APOE rs7412 was associated in our study with the Single Bright Invalid and Single Dim Invalid conditions, but in both cases it was associated in the opposite direction expected based on the literature regarding physiological impact of stress [APOE’s association with RT slope on the Single Bright Invalid condition is a replication of our finding in another sample using adult participants [There were two associations between the  disease . APOE rsf stress . Since bicipants .CHRNA4 had a single SNP (rs6090387) that was associated with two dim outcomes, Single Dim Invalid and Single Dim Valid. In contrast with APOE (where both luminances were affected), here both validities are affected. This implies that it is the luminance  that is giving some participants more difficulty as the task continues. We had anticipated that dim cues might give some participants more trouble because, while they are still clearly visible, they are not as salient and the bright cues [ght cues . The linSLC5A7 had two SNPs (rs3806536 and rs4676169) that were associated with the Neutral Bright outcome. Rs4676169 was also associated with the No Cue condition. SLC5A7 is a choline transporter involved in acetylcholine synthesis for cholinergic neurons [ neurons . As ment neurons –58, but  neurons –62.SNAP25 also had a SNP (rs6077690) that was significantly associated with two outcome measures, Single Dim Invalid and Single Dim Valid. This gene acts in the presynaptic plasma membrane during neural transmission and is involved in the regulation of neurotransmitter release . SNAP25 has been associated with ADHD and symptoms of ADHD, such as impulsivity [CHRNA4 and suggests that the relatively lower saliency might make this task progressively more difficult for some participants who are sensitive to luminance saliency in external cues. This seems to be the overall pattern in the cholinergic genes, that they are more sensitive to cue luminance such that dim cues  distinguish more between cholinergic genotypes.ulsivity –66. The DRD4, IGF2, and SLC6A3), all three were associated with the No Cue condition, two were associated with the Neutral Bright condition, and one each with the Single Bright Invalid and Single Dim Invalid conditions.Of the three significant genes associated with dopamine (DRD4 is a type-four dopamine receptor and has been associated with ADHD and novelty seeking [ seeking –73. Our  seeking –62. ThisIGF2 had one SNP (rs734351) associated with an outcome. Like DRD4, IGF2 is associated with the No Cue condition. The IGF2 gene is an imprinted gene . It is involved in growth and development generally, including cell differentiation of dopamine neurons during brain development [elopment . Even thSLC6A3 is also known as the dopamine transporter, DAT1. The SLC6A3 gene had six associations on the reflexive attention task. One of the findings (SNP rs2042449 with the No Cue condition) has an unknown risk allele. Our results are consistent with T as the risk allele (because those with the TT genotype get slower over the course of the task for this condition) or as consistent with heterozygous advantage (because heterozygotes get the fastest over the course of the task). The VNTR results are consistent with the direction of effects for sustained attention. However, the direction of effect for another SNP (rs2617605) appears to show opposite effects for our sustained attention task on the Neutral Bright and Single Bright Invalid conditions than those established for sustained attention. The two remaining associations (No Cue with rs2937639 and rs6350) are inconsistent with that established for sustained attention tasks. This is an important and useful finding because it indicates that reflexive attention might have fundamentally different associations than sustained attention.SNAP25 is associated with all three neurotransmitters we discuss. SNAP25 was associated with Single Dim Invalid and Single Dim Valid conditions. As such, it shares association with one other dopaminergic gene. SLC6A3 has one SNP (rs6350) associated with Single Dim Valid.As was mentioned above, Overall the pattern for dopamine is similar to that for acetylcholine, showing sensitivity to luminance. However, for dopamine it is bright cues that differentiate more between genotypes. It is notable that there were five significant associations with bright cues and only one significant association with dim cues for these genetic markers.BDNF and HTR4), one was associated with Neutral Bright and one with Single Dim Valid. The BDNF gene had two associations with RT slope outcomes for the Neutral Bright condition. The BDNF gene promotes differentiation, axonal growth, pathfinding, and dendritic growth during development. It is associated with the neurotransmitter serotonin, which is active during development [SLC5A7, each replicates the other result to confirm the importance of BDNF in reflexive attention. That a gene involved in brain development is associated a reflexive attention task in childhood suggests that there may be a somewhat inadequate serotonergic system in some children with attentional deficits.Of the two significant genes associated with serotonin (elopment . The risHTR4 had one SNP (rs1862345) that was significantly associated with slope for the Single Bright Invalid condition. HTR4 codes for a serotonin receptor. Reduced receptor number might decrease neural transmission even if there is otherwise enough serotonin available. However, somehow too many receptors is also nonideal. Perhaps too many receptors interfere with other actions necessary for neural transmission. Heterozygous disadvantage can happen when there is an optimal amount of a gene product (receptors) for performance and too much or too little can interfere with a particular outcome. One neurotransmitter with optimal level effects is dopamine [dopamine –62, but SNAP25 is associated with all three neurotransmitters we discuss. SNAP25 was associated with Single Dim Valid conditions as were CHRNA4 in the acetylcholine system, SLC6A3 in the dopamine system, and HTR4 in the serotonin system. SNAP25 was also associated with Single Dim Invalid. This seems to indicate that dim cue conditions are especially sensitive at detecting issues with neurotransmitter release.As was mentioned above, APOE (1 marker), BDNF (2 markers), CHRNA4 (1 marker), DRD4 (1 marker), IGF2 (1 marker), SLC5A7 (2 markers), SLC6A3 (5 markers), and SNAP25 (1 marker). The genes are associated with all three neurotransmitters we tested . All these neurotransmitters have been established to have association with sustained attention.Overall our results show that Neutral Bright cues and No Cue conditions (conditions which do not bias attention to one side or the other) distinguish well by genotype for genes associated with all three neurotransmitters. Conditions with dim cues also seem sensitive to genetic differences . Other patterns in the data suggest that there are different risk alleles for reflexive attention tasks than for sustained attention tasks and that there are optimal levels of availability of neurotransmitters (evidenced by heterozygous advantage). The genes that had significant markers on them were As with all genetic association studies, our findings will need replication. This is especially true because genetic association studies with reflexive attention tasks are rare. However, we have shown partial replication and extension of previous results using sustained attention to reflexive attention tasks. In some cased the direction of effects were the same as for disorders commonly associated with sustained attention deficits, but more often the results were in the opposite direction. This indicates the additional usefulness of reflexive attention tasks in studying attentional deficits. Studies should not be limited to sustained attention tasks when measuring attention. We have also contributed by making associations with SNPs for which the direction of effect was not known even though they are on genes associated with attention. This information is useful and warrants replication attempts. Ultimately, the present results add further evidence that genetics do contribute to slope variability in reflexive attention tasks. Further research into the genetics of attention has the potential to improve intervention for those with attentional deficits by targeting for intervention the specific types of tasks that give them trouble.


Pakistan has moved from a ‘low prevalence–high risk’ to a ‘concentrated epidemic’ state, yet the forcefulness required for managing this silent escalation of HIV infected numbers is not being highlighted, as it should be. A more comprehensive review of the national strategy for HIV/AIDS would necessitate a system's thinking. For this purpose, the WHO's Health Systems Building Blocks have been discussed to analyse whether this framework can be employed to take some corrective measures. An extensive literature review in this regard helps to understand that the service delivery has to be responsive, but skilled human resources, a robust information system, an uninterrupted supplies and use of latest technology, adequate financing, and above all good governance at operational level are essential ingredients, which call for re-orienting the national programme today. Lack of coordination, capacity, and interventions with questionable sustainability pave a perilous path. Hitherto, the issue can be addressed by involving stakeholders from all levels of the society and managing the void between policy and implementation. Furthermore, interventions that focus on the long-term future are imperative to combat the menace threatening human lives. From the emergence of the first case in 1987 to an alarming toll of more than 83,000 HIV/AIDS cases, Pakistan has moved to the category of a concentrated epidemic country . In spitTo build our main set of arguments, we have chosen the WHO's Health Systems Building Blocks to elaborate the present situation with regards to the response to the HIV epidemic in Pakistan. Each building block is discussed separately in this article based on the information available in the existing literature. To explain the situation according to each building block, a literature search was conducted over the internet, using nomenclature of the building blocks, that is, service delivery, health workforce, information system, drugs-supplies-technologies, financing, and leadership, and governance in HIV/AIDS sector. The online library of Pakistan's National AIDS Control Programme and websites of The Global Fund to Combat AIDS, Tuberculosis and Malaria and the World Health Organization were used for our literature search. We consulted Google Scholar, for the same Medical Subject Headings (MeSH) terms, and the full texts of articles of our choice were retrieved from Medline/PubMed, where available.Like any other developing country, in Pakistan, first level care is mostly sought in the private sector, because of distrust in the quality of care in the government system, limited working hours, shortage of trained human resources, and dearth of supplies . A meagrHIV treatment is available at designated anti retroviral therapy (ART) centres. Nevertheless, the treatment, care, and support facilities are available in the large metropolitans only . AccordiThe health workforce for HIV/AIDS is not only deficient in number but also lacks the required set of skills needed for dealing with HIV/AIDS cases .The need for future planning regarding an improved health workforce is strong because the skill set needs to be upgraded with the changing patterns that HIV spreads in the country. There must be a continuing medical education programme for postgraduate trainees as well as the general practitioners to keep them abreast with the latest developments in the HIV/AIDS sector. More incentives for the workers would definitely keep their motivation alive for serving the difficult cases. Rural-based service rotations can be made a policy point so as to cater for the unequal distribution of doctors and nurses at the primary health care level.The public sector health management information system (HMIS) system lacks completeness, authenticity and therefore needs complete revamping. Moreover, the information from the vertical programmes like HIV/AIDS needs a meaningful integration in HMIS for timely decision making .The individuals coming in for HIV testing and for registered cases seeking treatment from public facilities are given free treatment. However, at times, a serious shortage of anti retroviral vaccine (ARV) is observed because of poor future planning. Procurement guidelines, schedules and supply chain must be revisited and adapted in the wake of the changing burden of disease and transition of roles between the national and provincial programme offices.In Pakistan, the HIV/AIDS programme has always depended on the donors and extra mural funding. The bilateral and multilateral donors and other development partners have invested to address the HIV threat in Pakistan. The Global Fund's total pledge to HIV/AIDS in Pakistan is US$ 37,523,500. However, the grant performance has been rated as inadequate and did not meet the expectations . In all Leadership of the national programme has been a complex affair. Political transition, administrative decentralisation, and donor dependency have their own implications. Governance reflects certain weaknesses such as shortage of supplies, irregularities in the use of funds, and violation of rules in appointments and procurements . BesidesThe health system in Pakistan reflects a definite lag in terms of achieving the millennium development goals (MDGs) of maternal and child health as well as of tuberculosis, malaria, and HIV/AIDS. It would be critical to carry out a thorough stocktake to analyse the performance of the health system of Pakistan, in light of planned and achieved targets of HIV/AIDS. Such a gap analysis will give an account of gaps and flaws in the system, responsible for the current state of affairs. This is an opportune time to carry out this exercise, because the ongoing reforms will have the chance to look into deficiencies of all building blocks of the health system . The WHOAll stakeholders working to win the battle against HIV/AIDS must be convinced to work and employ health system strengthening building blocks if desired results are to be achieved. The national programme of the government, NGOs, and the donors must invest their energies to review and strengthen all aspects from financing to governance, and from service delivery to human resources, with evidence-based decision making, using authentic and reliable information systems.