Antibiotic innovation has dwindled to dangerously low levels in the past 30 years. Since resistance continues to evolve, this innovation deficit can have perilous consequences on patients. A number of new incentives have been suggested to stimulate greater antibacterial drug innovation. To design effective solutions, a greater understanding is needed of actual antibiotic discovery and development costs and timelines. Small and medium-sized enterprises (SMEs) undertake most discovery and early phase development for antibiotics and other drugs. This paper attempts to gather a better understanding of SMEs’ targets, costs, and durations related to discovery and early phase development of antibacterial therapies.n = 44) were subsequently sent a survey to gather more data regarding their areas of activity, completed and expected development costs and timelines, and business models.DRIVE-AB, a project focused on developing new economic incentives to stimulate antibacterial innovation, held a European stakeholder meeting in February 2015. All SMEs invited to this meeting  costs. The duration ranges reported for completing each phase of R&D are highly variable when compared to previously published general pharmaceutical innovation average durations. However, our sample population is small and may not be fully representative of all relevant antibiotic SMEs.The data collected by this study provide important insights and estimates about R&D in European SMEs focusing on antibiotics, which can be combined with other data to design incentives to stimulate antibacterial innovation. The variation implies that costs and durations are difficult to generalize due to the unique characteristics of each antibiotic project and depend on individual business strategies and circumstances.The online version of this article (10.1186/s40545-018-0135-0) contains supplementary material, which is available to authorized users. The world is facing an emerging threat of greater antibiotic resistance . New antwww.drive-ab.eu), a consortium of 16 public sector partners and seven pharmaceutical companies, which aimed to transform the way policymakers stimulate innovation, sustainable use and equitable access of novel antibacterial products to meet public health needs. This article is a part of DRIVE-AB’s research efforts.Antibacterial innovation is receiving significant political attention of late, including in the G7 and G20 groups of countries , the WorA variety of economic incentives have been proposed to stimulate antibacterial drug innovation –15. SincActual pharmaceutical R&D costs are deemed highly confidential and controversial. Few researchers have been allowed access to this type of data, with the one main exception of DiMasi and colleagues at Tufts University, whose results are based upon data from ten, large pharmaceutical companies focusing on a range of therapeutic areas . The stuThis article is meant to shed some light on the targets, costs and durations of early phase development  for antibiotic innovation (see Table Forty-four (44) European-based SMEs were invited to attend a stakeholder meeting in London in February 2015, with a purpose to understand the environment in which SMEs operate, their motivations, and the challenges they face in undertaking antibiotic R&D. The list of companies and contacts was gathered through expert advice and personal contacts of all known European SMEs with at least one antibacterial project in their pipelines. Out of the 44 SMEs invited, representatives of twenty-six (26) companies attended the meeting. The companies varied in size, from virtual companies with no full-time employees to those with dozens of employees. The attendees were divided into four groups with rapporteurs assigned to each group. Each group followed a discussion guide which included challenges faced by antibacterial drug-focused SMEs, financial barriers to investment, the role of SMEs in relation to other R&D organizations, and brainstorming on potential incentives to address SME challenges. Findings from the group work were then discussed in plenary. A final meeting report has been produced which includes the discussion questions, as well as the list of the attending SMEs .A survey  had no revenues in 2014. 68% of the respondents (n = 17) focused on 1 to 3 internal antibacterial projects. In addition, 40% of the respondents (n = 10) outsourced more than half of their R&D budget to external organizations.Twenty-five (25) SMEs responded to our survey (a response rate of 57%). These can be classified mostly as small companies since only one company has more than 100 employees. 54% of the respondents (n = 20) identified their own research as the source of their lead antibacterial project. In a separate question, 20% (n = 4) reported that they are spin-offs from universities or research institutes and another 20% (n = 4) spin-offs from a large, multinational pharmaceutical companies.The stakeholder meeting identified three main sources of discovery of companies’ antibacterial projects, either they were discovered: (1) in an academic setting which led to the establishment of a spin-off company, (2) independently by an expert in this field who subsequently formed a new company, or (3) in a large pharmaceutical company and subsequently spun-off as an SME. 80% of the survey respondents (n = 19) perform R&D for small molecules, and 44% (n = 11) focus on antibodies, adjunctive antibacterial technologies including phage-based therapies and preventive vaccines.  Most companies focus solely on human health, but 28% (n = 5) also target animal health and/or environmental issues. The overwhelming majority of survey respondents claimed that they are involved in research and development of novel products, with 56% (n = 14) pursuing a novel class and 20% (n = 5) a novel mode of action.Companies at the stakeholder meeting expressed that they entered the antibacterial market because there are significant public health opportunities in new antibacterial products with little competition. Several of these stakeholders also claimed that these opportunities are linked to significant unmet public health needs. The research focus of the survey respondents is largely small molecule development, i.e., traditional antibiotics. 76% (n = 11) of respondents stated that the focus of their main antibacterial project is a narrow-spectrum target and 36% (n = 9) a pathogen-specific approach. 80% (n = 20) of respondents reported their main antibacterial project targets, including Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, Neisseria gonorrhoeae, Clostridium difficile, and/or Staphylococcus aureus.Participants at the stakeholder meeting agreed that the increasing resistance problem is the primary market opportunity for antibacterial R&D, and SMEs aim, therefore, to meet therapeutic needs caused by emerging resistance against existing antibiotics. 44% (n = 17) of respondents expressed hope to be acquired, 63% (n = 15) expressed hope to out-license their products, and only 17% (n = 4) would consider commercializing their products on their own. This in turn translates into their expectations on the extent of their development work. 36% (n = 9) of respondents aimed to complete Phase II clinical trials before out-licensing or selling their main antibacterial projects, whereas 24% (n = 6) aimed to complete Phase I clinical trials. And 24% (n = 6) aimed to out-license or sell prior to clinical trials.SMEs focused on pharmaceutical innovation typically perform discovery and early phase development work . SMEs at the stakeholder meeting stated that they struggled to find an exit strategy or pathways to commercialize their products due to the paucity of large, pharmaceutical companies actively pursuing R&D on antibacterial products. Many did not see a realistic way to commercialize products by themselves, but were considering taking on this role due to the few commercialization options. When asked to select among multiple exit strategies, 71%  in order to report similar activities. Figure The following development costs and timelines relate only to those who reported performing R&D on small molecules n = 9 in ordeEight SMEs have reported that they have completed lead compound identification. This may seem contradictory since 18 companies have reported that they are currently beyond lead compound identification. We can only presume that these ten companies either chose not to report the data or that they acquired the main antibacterial project after lead compound identification had already been performed. One company, at the time of the survey, was currently performing lead compound identification.n = 3) to more than € 1 million (n = 3)  and estimated costs for lead compound optimization.Among the four companies that have completed this phase, one took less than a year, another took one to two years, and the remaining two took two to four years. Amongst the seven companies, at the time of the survey, currently performing this phase, one company expects it to take less than six months, another predicts it will take six months to one year, four expect it to take one to two years, and the remaining company expects two to four years.Three out of the four companies that have completed the phase reported the costs incurred. One reported costs less than € 1 million and the other two companies between € 1–5 million. Among the eight companies with expected costs, six expected that the phase will cost between € 1–5 million, whereas two others estimated less than € 1 million. See Fig. Out of the twelve companies in total reporting for lead compound optimization, seven claimed that the main antibacterial project represented a novel class and four claimed a novel mode of action. (The remaining company was performing R&D on a known class.) However, neither duration nor cost seemed to be influenced by novelty, meaning that the products described as novel were spread across all cost and duration ranges. The majority of SMEs have spent (or plan to spend) from € 1 to 5 million on lead compound optimization which can take as short as 6 months or as long as 4 years.Four companies reported that they have completed preclinical testing. Five companies reported, at the time of the survey, that they were currently performing preclinical testing. Seven companies, at the time of the survey, were at the phase of lead compound optimization  and therefore estimated the costs for preclinical testing.Among the four companies that have completed the phase, only three reported time durations. All took from one to two years. Amongst the five companies, at the time of the survey, currently performing the phase, one expects it to take six months to one year, and the remaining four expect it to take one to two years.Amongst the four companies that have completed the phase, one reported costs less than € 1 million and the remaining three between € 1–5 million. Among the twelve companies with expected costs , two expect that the phase will cost less than € 1 million, eight between € 1–5 million, and two between € 5–10 million. See Fig. Out of the 16 companies in total reporting for preclinical testing, nine claimed that the main antibacterial project represented a novel class and three claimed a novel mode of action. (The remaining four companies are performing R&D on known classes.) However, neither duration nor cost seemed to have been influenced by novelty, meaning that the novel products were generally spread across all cost and duration ranges. Both two out of three of the least expensive projects (less than € 1 million) and the two most expensive projects (estimated to cost € 5–10 million) represent novel classes. The shortest duration (6 months to one year) was to complete preclinical testing for a project related to a known class. The majority of SMEs have spent (or plan to spend) from between € 1 and 5 million on preclinical testing which can take approximately one to two years.Six companies reported that they have completed Phase I clinical trials. Two companies reported, at the time of the survey, that they were currently performing Phase I clinical trials. Five companies, at the time of the survey, were performing the previous R&D phase  and estimated costs for Phase I clinical trials.Among the six companies that have completed the phase, three reported that it took six months to a year to complete Phase I clinical trials, two reported between one to two years, with the remaining reporting five or more years. Amongst the two companies, at the time of the survey, currently performing the phase, one expects it to take less than six months and the other six months to a year.Among the six companies that have completed the phase, two reported costs less than € 1 million, three between € 1–5 million, and the remaining between € 10–15 million. Among the seven companies with expected costs, one expects that the phase will cost less than € 1 million, four between € 1–5 million, and two between € 5–10 million. See Fig. Out of the 13 companies in total reporting for Phase I clinical trials, seven claimed that the main antibacterial project represented a novel class and two claimed a novel mode of action. (The remaining four represented R&D on known classes.) However, neither duration nor cost seemed to have been influenced by novelty, meaning that the products described as novel were spread across all cost and duration ranges. The one company that reported a duration of five years or more is developing a novel class. The majority of SMEs have spent (or plan to spend) from € 1 to 10 million on Phase I clinical trials which can take six months to two years.Only one company has completed Phase II clinical trials. In order to safeguard the anonymity of results since few European antibacterial-related SMEs have completed Phase II clinical trials we have combined the completed and expected figures for Phase II. Three companies, at the time of the survey, were performing Phase II clinical trials, and two companies were performing Phase I clinical trials and reported the estimated costs for Phase II as well.Four companies reported duration data, two reported that it took one to two years to complete Phase II clinical trials and the other two reported between two to four years. Among the six companies reported cost data, one reported costs less than € 1 million, one between € 1–5 million, two between € 10–20 million, and the remaining one more than €20 million Fig. .Fig. 6OuOut of the six companies in total reporting for Phase II clinical trials, four claimed that the main antibacterial project represented a novel class and one claimed a novel mode of action. (The remaining company is performing R&D on a known class.) However, neither duration nor cost seemed to have been influenced by novelty, meaning that the products described as novel were generally spread across all cost and duration ranges with the notable exception that the most expensive assertion (more than €20 million) was related to trials for a known class. The majority of SMEs have spent (or plan to spend) from € 1 to 20 million on Phase II clinical trials which can take one to four years.The results from our stakeholder meeting and survey provided data regarding development targets, development costs and timelines of European antibacterial-focused SMEs. These may not be a full representation of antibacterial SMEs in general since the sample population is small, and it becomes even smaller when the data are further sub-divided by R&D phase. Additionally, our findings report both actual and expected out-of-pocket costs within fairly broad ranges (as indicated in each of the previous figures). Expected costs may change over time. However, the results provided important insights into the therapeutic targets, R&D costs and R&D phase durations of European SMEs operating in antibiotic development.DRIVE-AB and other initiatives need estimation of cost and duration to be made in order to effectively calculate adequate rewards. An “adequate” reward is one where the publicly-sponsored reward will generate a positive return on investment  for the innovator without the public sector over-paying. Based on our results we propose the ranges in Table In order to give a perspective regarding our proposed minimum and maximum costs per R&D phase we include two other studies , 22 whicOur findings suggest that SMEs expect to perform antibacterial drug development less expensively than large pharmaceutical companies. One interpretation is that SMEs are leaner in structure and more cost-effective in their R&D activities. An alternative explanation may be that SMEs focus mainly on the essential studies that are minimally required by regulatory agencies for entry into limited Phase 2 studies and that they conduct additional studies at later time points. Total costs may be higher than those reported by SMEs here as the company that intends to commercialize the antibiotic may need to back-fill missing data to get licensure. Larger companies may include additional supporting studies or study variables to increase confidence in study results and reduce future risk of failure while a smaller company might be willing to take greater risk while anticipating an exit prior to regulatory approval. Recent changes in the regulatory landscape have significantly influenced costs and duration of later phases of clinical development with the option of abbreviated special pathways with smaller trial sizes. SMEs may also be anticipating these cost savings from regulatory changes; however, these should mostly be related to Phase III clinical trials which are not included in our study. Lastly, as stated clearly throughout the paper, a large percentage of the data is estimated amounts which may vary when faced with real-world obstacles. While the sample companies are inexperienced with performing more advanced development work, at least some SME executives have substantial drug development experience from previous employment.Table Our duration ranges demonstrate a great deal of variation. The range generally contains the estimates from both the general pharmaceutical innovation and Sertkaya et al., with the exception of Phase I clinical trials. Indeed, in each R&D stage, the duration and costs reported by the surveyed companies vary by a factor of 10 or even more , medicines regulatory authorities, SMEs, national research funding agencies, academic research institutions, and more. Although principally European in focus, DRIVE-AB actively engaged stakeholders globally to ensure that its recommendations can be integrated in a broader context to ensure sustainable access to efficacious antibiotics and ultimately combatting resistance.DRIVE-AB performed a computer simulation on different innovation incentives and combined several estimates of costs and durations such as those reviewed in this paper in order to calculate potential profits. It was valuable to have direct data from SMEs in order to develop realistic reward models. Despite the abovementioned limitations, the findings in this paper provide further insights that can help devising more precise policy tools for simulating pharmaceutical innovation taking into account their costs and durations. Similar research regarding SMEs outside of Europe would be beneficial.Additional file 1:Survey for SMEs. (PDF 447 kb)


EditorsA H SparkesInternational Society of Feline Medicine, Veterinary Division of International Cat Care, High Street, Tisbury, Wiltshire SP3 6LD, UKandy@icatcare.orgEmail: M ScherkcatsINK, Vancouver, BC, Canada V5N 4Z4hypurr@sagepub.co.ukhypurr@aol.comEmail: Editorial TeamC Bessant (Executive Editor)M Melling (Managing Editor)A Tansley (Assistant Editor)International Society of Feline Medicine, Veterinary Division of International Cat Care, High Street, Tisbury, Wiltshire SP3 6LD, UKjfms@icatcare.org; jfmsclinicalpractice@icatcare.orgEmail:


Cricket is one of the world’s most popular sports, followed by hundreds of millions of people. It can be dangerous, played with a hard ball flying at great velocities, and accidents have occasionally been fatal. Traditionally, cricket has been played during the day, using a dark red ball. Since the late 1970s, a shorter form of one-day cricket has been played both during the day and at night under floodlights. To overcome visibility issues, one-day cricket uses a white ball, and players wear coloured clothing. There is now a desire to play a traditional form of cricket during the day and at night, using a ‘pink’ ball while players wear white clothing. Concerns regarding visibility, and player and umpire safety, have been raised in this context. Here, we report that these concerns have a sound basis. The desire to play a traditional form of cricket, during the day and at night under floodlights, creates a challenging visual environment. During play, the dominant light source undergoes dramatic changes, in intensity and composition. During traditional daytime playing hours, in optimal viewing conditions, the sun can be said to appear yellow due to the dominant wavelength of light it emits . As the Changes in the composition of light can alter the appearance of coloured objects, due to changes in the relative intensities of light reflected from different surfaces. The intensity of light reflected from a surface that looks red will, for instance, tend to become proportionally greater at sunset than at midday, relative to a surface that looks yellow or green. This reflectance ratio would change once more as the dominant light source shifts from being the setting sun to being the stadium floodlights (at night). When considering what type of impact these changes might have on player and umpire safety, one should be aware that human motion perception is driven predominantly by encoded differences in brightness. Hence, it is possible for an object to be visible, because it has a distinctive colour relative to its background, without people being able to accurately judge its speed, because it is nearly equally bright relative to the background .white sightscreen and coloured stands. Regularly sampled balls included a traditional dark-red cricket ball, a white one-day cricket ball and a pink day-night cricket ball  first class day-night cricket match, played at the GABBA (a first-class ground) in Brisbane, Australia, using a recently developed pink cricket ball. We were able to take readings of the intensity of light reflecting off several surfaces on the second day/night of play. We were able to take some readings regularly during play, whereas others could only be taken intermittently, during breaks in play. We split readings between background surfaces, against which players and umpires need to see a ball, and the cricket balls themselves. Regularly sampled backgrounds included the sky, field (adjacent to the boundary rope), the darker than all pertinent backgrounds . Similarly, white ball readings suggest it was reliably brighter than all pertinent backgrounds . The pink ball, however, changes the polarity of its luminance contrast relative to pertinent backgrounds, including the sky, outfield and grand stands, during this period.We have depicted luminance readings taken from regularly sampled balls and background surfaces in To highlight pink ball polarity reversals, in Although we were able to sample the match ball and pitch less often, recordings suggest that these too were subject to a luminance contrast polarity reversal during play. In We have focused on the luminance contrasts of balls relative to pertinent backgrounds, as opposed to colour contrast. We suspect many people will be unaware that colour contrast contributes strongly to visibility but has little impact on speed perception (which is instead governed by luminance contrast). We will, however, mention one aspect of visibility. At dusk, when viewed against the grandstands during play, we found it entirely impossible to see a fast moving pink ball. This period coincided with a time at which the ball was nearly equal in average physical luminance relative to the stands, but in addition to this the stands were multi-coloured, and thus served as an effective mask for the fast moving pink ball. We suspect players and umpires would have experienced similar difficulty.It remains to be seen how far our observations would generalize, and factors other than physical reflectances should be considered. For instance, light intensity reduces near sunset, so the composition of photoreceptors in the human eye used to encode input undergoes change, and this too can impact the appearance of coloured objects and speed perception . The relWith these caveats in mind, we do, however, believe our data speak to concerns that will generalize. First, the pink ball will inevitably undergo a change in contrast polarity relative to the sky. So, for a period, around dusk, players might experience difficulty in judging ball speed if they must see a pink ball moving with the sky as a backdrop. While it might not always undergo a reversal in contrast polarity relative to the field and pitch, we believe the change in the composition of light about sunset will inevitably lessen ball contrast against these surfaces at sunset. Behaviourally, this could result in the ball looking ‘blurred’ and in participants being slow to react. We believe these observations accord with concerns raised by players and umpires, and so encourage caution when considering changes to policies governing playing conditions.In the interest of safety, it would be sensible to take reasonable precautions to ensure cricket is played in optimal visual conditions. Our data suggest visual conditions for a pink ball are degraded at sunset. This could be resolved by scheduling a sufficient break in play centred on sunset. Alternatively, a white-coloured ball sufficiently robust to withstand the rigours of test cricket might be developed. A third possibility is that glasses with appropriately coloured lenses could be used, to enhance the pink ball brightness relative to other surfaces.


A step in the correct direction.............Second prize in journal category by‘The Federation of Indian Publishers’A very happy new year to all and hope this year ushers in the best health and wishes for all. It gives me immense pleasure to announce that our journal, International Journal of Clinical Pediatric Dentistry (IJCPD) has won the second prize in Journal category printed and published in India by ’The Federation of Indian Publishers’. It is a moment of immense pride for the entire team, editorial staff, Jaypee group, editorial board, reviewers and most importantly the authors of articles who have always trusted us and shown faith in us for their scientific manuscripts. I assure you that this is just another milestone for us and the journey will continue as we have miles to go.Expansion is the essence of time and we at IJCPD believe that it is always more advisable to have a large number of contributors as it increases our scientific acumen and also the journal’s outreach among society. I whole heartedly welcome Drs Satish Vishwanathaiah and Ravi GR who have joined us as Assistant Editors. International Journal of Clinical Pediatric Dentistry is also pleased to welcome new members Drs Rupinder Bhatia, Nikhil Srivastava, Srinivas Namineni, Sukhdeep Singh, Arathi Rao, Mandeep Singh, Neeraj Gugnani, Vijayprakash Mathur, Ameet J Kurthukoti, Anshula Deshpande and Puneet Goenka to its editorial board and I am sure their valuable contribution and knowledge will help the journal to achieve its further growth.International Journal of Clinical Pediatric Dentistry is one of the few journals in the world which publishes a preliminary list of articles on its website which are going to appear in subsequent issues (until 2019). We are also in the process of making these articles available online for the authors’ use very soon. We pridely transgress the information of having a transparent and time-bound review process so that waiting time for article decisions is not prolonged for the authors and we would improvise further in future to reduce the waiting time post-acceptance by increasing the number of articles in each issue or increase the number of issues in a year. While we work to make this journal more efficient and approachable, I, once again thank all for their valuable support and love and hope that you will continue to trust us for your scientific publications.Nikhil Marwah MDSEditor-in-Chief, International Journal of Clinical Pediatric DentistryAssociate Editor, JMGUMSTProfessor and Head, Department of Pediatric DentistryMahatma Gandhi Dental College, MGUMSTJaipur, Rajasthan, India


Porous starch granules (PSGs) with various pores and cavity sizes were prepared by amylolysis enzymes. The greatest hydrolysis rate on corn starch granule was observed with α-amylase, followed by gluco- and β-amylases. Temperature increase enhanced glucoamylase reaction rate more drastically than other enzyme treatments. Final hydrolysis level with glucoamylase reached to 66.9%, close to 67.5% of α-amylolysis. The α-amylase-treated PSGs displayed the greatest pore size and ratio of cavity-to-granule diameters. Gelatinization onset temperatures of PSGs increased to 72.1 (α-), 68.7 (β-), and 68.1°C (gluco-amylolysis) after 8 h; enthalpy changes of β- and gluco-amylase-treated PSGs increased to 13.4, and 13.1 J/g but α-amylase-treated one showed slightly reduced value of 8.5 J/g. Water holding capacities of PSGs were 209.7 (α-), 94.6 (β-), and 133.8% (gluco-amylolysis), and the untreated control had 89.1%; oil holding capacities of them showed 304.5, 182.7, and 211.5%, respectively, while the untreated control had 161.8%. Thus, enzyme types and their reaction conditions can be applied to generate desirable cavity and pore sizes in starch granules. This biocatalytic approach could contribute to develop tailor-made PSGs with distinct internal structure for specific uses in wide range of food, pharmaceutical and other industrial applications. Biopolymers are widely used as encapsulated agents and loading-carrier materials in food, agricultural, and pharmaceutical area. Among all natural available biopolymers, starch is the most commonly applied in the biomaterial fields due to its beneficial advantages such as low-cost, low-toxicity, biodegradability, and renewability . DespitePorous starch has been interested because it has high surface area and accessible inner empty space. These properties can be applied in food, cosmetics, and other related industries as absorbents as well controlled delivery materials . Porous For the enzymatic production of porous starch granule, α-, β-, and gluco-amylases, are the most common enzymes for structural modification of starch granule. α-Amylase can hydrolyze the internal α-1,4-glucosidic bonds of starch molecules with endo-hydrolytic activities except for those near the α-1,6 branching points. Basically, α-amylolysis occurs at any α-1,4-linkages in a random manner, resulting in wide pores on starch granules. β-Amylase is an exo-type enzyme that removes maltose units sequentially from non-reducing ends and stop the reaction near the α-1,6 branching points , 15. GluMany researchers studied the formation of pores on starch granules by amylolytic enzymes and investigated application of PSGs products as encapsulation or carrier agent , 13, 20.Aspergillus niger (Optidex L-400), β-amylase from barley , and α-amylase from Bacillus lichemiformis (Spezyme Xtra), were obtained from the Dupont™ Genencor® Science . Soybean oil was purchased from commercial products in a local market. Other reagent-grade chemicals were purchased from Sigma-Aldrich Chemical Co. .Normal corn starch (NCS) was provided from Ingredion Inc. , and it was used as a granule starch to prepare PSGs. Three different types of starch hydrolyzing enzymes, glucoamylase from One unit of hydrolyzing enzymes was determined as the amount of enzyme that catalyzes the production of 1 μmol of reducing sugars per minute. Individual enzyme assays were carried out in 50 mM sodium acetate buffer  containing 1 mg/mL of soluble starch  at 40°C for 10 min. After the reaction, the amount of released reducing sugar was measured by the dinitrosalicylic acid (DNS) method, using glucose for glucoamylase activity or maltose as a standard for α- and β-amylases , 22.g) at 4°C for 20 min, and the precipitated- modified starch pallet was washed twice with deionized water. The slurry was freeze-dried, and then the dried-sample was passed through a 100-mesh sieve. The sieved-product was stored in a desiccator at room temperature [Enzymatic reaction for producing the PSGs was carried out in 50 mM sodium acetate buffer  with 5% (w/v) of NCS. The individual hydrolyzing enzyme (800 U/g of starch) was added to the substrate mixture, and then the reaction was carried out at 30 or 40°C up to 8 h. After the reaction, 0.1 M NaOH was added to stop the enzyme activity. The dispersion was centrifuged ; and m is the molar mass of d-glucose in supernatant after hydrolysis reaction (180 g/mol), respectivelyTo determine the hydrolysis degree of PSGs, the diluted supernatant and DNS solution were mixed to ratio of 1:1. The mixture was kept in a boiling water bath for 5 min and was cooled in ice bath. The absorbance was measured at 575 nm using a UV/vis spectrophotometer . The degree of hydrolysis was determined using the following equation:The morphological properties of native corn starch and PSGs were observed by a field-emission scanning electron microscope . The samples were coated with platinum by a sputter coater  and were observed at 15,000× magnification under an accelerating voltage of 3 kV. Surface pore diameters of starch granule were measured, and the diameters of the longest axes in each of 5 pores within an image for a total of 5 images per sample were quantified by using the ImageJ software .Merbromin-treated starch granules  were plaThermal properties of PSGs were analyzed by using a differential scanning calorimeter . The starch sample (5 mg) was exactly weighed in an aluminum pan, and deionized water (15 mg) was added. This starch dispersion was equilibrated at ambient temperature for 24 h. The sealed sample was scanned from 20 to 120°C at a heating rate of 5°C/min, using an empty pan as a reference .dry basis) was suspended in distilled water (4 mL) and shaken vigorously to fully disperse the starch. The suspended starch solution was allowed to stand for 10 min at ambient temperature. After the 10-min suspending period, the dispersion was centrifuged , and the sediment was weighed. Water holding capacity of PSGs was calculated according to the following equation:W0 is the initial weight (g) of starch sample before treatment; W1 is the final weight (g) of starch sample after treatment. The analysis result was presented as the mean value of three replications [To measure the water holding capacity, starch  of starch sample before treatment, and W1 is the is the final weight (g) of starch sample after treatment. The results presented are the mean values of three replications [To measure the oil holding capacity, soybean oil (6 mL) was added to sample (0.5 g) in a graduated 50 mL centrifuge tube. The tube was vortexed for 1 min, left for 30 min and centrifuged for 25 min at 3000 × ications .® SPSS® Statistics for Windows . Statistical significance in the difference among the values was evaluated by Tukey’s test at p < 0.05.All statistical analyses were performed by using IBMv0) of α-amylase (5.8×10−3 s-1) on native starch granule was faster than glucoamylase (1.1×10−3 s-1) and β-amylase (7.8×10−5 s-1) at 40°C  was similar to the resulted at 40°C within 2 h (33.0%) by α-amylase treatment.v0 of glucoamylase more drastically than other two enzymes, and eventually the degree of hydrolysis by glucoamylase reached the plateau (66.88%) of which the level was very close to that of α-amylase (67.46%) after 12 h. The amount of released glucose after 4-h α-amylase treatment was similar to that of 8-h glucoamylase treatment at 40°C. It was caused by the difference in reaction pattern between α-amylase and glucoamylase. α-Amylase is an endo-type enzyme that can randomly hydrolyzed α-1,4 glycosidic bonds of starch, and glucoamylase is an exo-type enzyme that can not only hydrolyze α-1,4 glycosidic bonds but also α-1,6 glycosidic bonds [v0 of α-amylase was faster.The temperature effect on the hydrolysis level of glucoamylase was even greater, thus it produced more than three times larger amount of reducing sugars at 8 h and thereafter. As a result, the increase of the reaction temperature by 10°C enhanced the ic bonds . Thus, gβ-Amylolysis for 12 h showed that the starch granules were hydrolyzed in 1.7 and 2.8% of degrees at 30 and 40°C, respectively. Compared to these α-amylase and glucoamylase enzymes, β-amylase hydrolyzed the starch granules very slowly, and it was unable to destruct native starch granule effectively. Also, it was well reported that β-amylase hydrolyzed very slowly on all the starches over α-amylase . AlthougIn the FE-SEM analysis, α-amylase-treated starch granules showed relatively fewer number of large pores with many cracks whereas glucoamylase-treated ones had lots of small pores under the same conditions . The sizWhen the same degrees of starch hydrolysis were obtained from an enzyme treatment with different reaction temperatures and times, it was noticed that the size and morphology of pores on surface of starch granules were almost identical. Thus, amylolysis at 40°C for 2–4 h would be better applicable than the treatment at 30°C for 8 h. In this observation, it was affirmatively supported by the previous research  that theThe internal structure of enzyme-treated starch granules at 40°C during different reaction time internals was visualized by means of the confocal microscopic method . When stc) over granule diameter (Dg) of PSGs produced by amylolytic enzymes at 40°C was determined from different reaction time points  of starch granule in the presence of water has been known to be affected by the structure of crystalline region, which corresponds to the external chain of amylopectin molecules formed with the double helical structure [o = 66.7°C) and enthalpy change (ΔHgel = 10.6 J/g) of NCS, enzymatic digestion of native starch granule caused to increase To by at least 0.7°C from 2-h gluco-amylolysis and up to 5.3°C from 8-h α-amylolysis  of starch granules was also observed by all the enzyme treatments in this melting phenomenon but there was no significant difference among the enzyme-treated samples. The endo-type enzyme might be much easier to access and hydrolyze both amorphous and imperfect crystalline regions inside starch granule in the diffusive mode, once the enzyme made complete holes in the centripetal mode [ΔHgel over the reaction time [ΔHgel within initial 2 h of exo-amylolysis.From our enzymatic hydrolysis study, it was shown that endo-type α-amylase increased the Ttal mode . As a reion time . Becauseion time , and thuIn an effort to utilize the porous structure of starch granule as a delivery system of nutrients or drugs, its loading capacity was evaluated by applying two different liquids, water and oil. When enzymatic hydrolysis was pursued at 40°C, the reaction time influenced on water holding capacity (WHC) of starch granules . As the Oil holding capacity (OHC) of starch granules was also affected by enzymatic hydrolysis at 40°C . As a coIn this study, different types of PSGs were prepared by amylolytic enzymes such as α-amylase, β-amylase and glucoamylase which were able to generate various pores and cavity sizes on the surface. Unique structural and biochemical properties of PSGs can be designed and produced by different amylolytic enzyme treatments. When we utilized the internal cavity and expended surface area of PSGs, generated by these enzyme treatments, the holding capacity of water and oil was significantly increased with optimal porous structure production. Thus, different types of α-amylolytic enzymes and their various reaction conditions can be applied to produce desirable cavity and pore sizes in raw starch granules. This biocatalytic approach could apply to design tailor-made PSGs with distinct internal structure for specific uses as a novel carbohydrate-based delivery system or encapsulant in wide range of food, pharmaceutical and other industrial applications.


CLINICS 2017;72(5):294-304Retraction requested by the Editor of CLINICS in agreement with the authorsRetraction type: Redundant publicationhttp://www.tncjournal.com/EN/10.18679/CN11-6030/R.2017.003).Comments: The same article was published almost simultaneously in the journal TRANSLATIONAL NEUROSCIENCE AND CLINICS with a different title: “Factors affecting the prognosis of children with medulloblastoma: A single institution retrospective analysis of 40 cases” (All articles submitted to CLINICS undergo strict quality control and are checked using iThenticate software before being submitted for peer review and before acceptance. The retracted article was checked on September 26, 2016, and February 24, 2017. Unfortunately, the article was published in TRANSLATIONAL NEUROSCIENCE AND CLINICS in May 2017, so there was no way of acknowledging the publication during the evaluation process. The authors submitted all required copyright transfer documents stating that the manuscript had not been submitted to any other journal.“It was an unintentional mistake caused by carelessness. After acceptance of our article in CLINICS, we attended a seminar. TRANSLATIONAL NEUROSCIENCE AND CLINICS indexed our paper after the seminar, and we knew nothing about this at that time. We misunderstood and thought that this would not influence the publication of our paper in CLINICS as TRANSLATIONAL NEUROSCIENCE AND CLINICS is a non-SCI journal, and the paper was indexed only internally.”The authors stated the following:


The Brazilian Journal of Cardiovascular Surgery (BJCVS) is grateful for the reviewers,listed below, which collaborated in this edition. Without their work would be impossibleto keep the high scientific standard of our journal.Bruno RochaCarla TanamatiEdmo GabrielEdward GologorskyGuilherme SucciHenrique MuradJoão Carlos Ferreira LealJosé Glauco Lobo FilhoMagaly Arrais dos SantosMauro Paes Leme De SáNelson HossnePaulo Roberto EvoraRui M. S. AlmeidaSergio Francisco dos Santos Junior


Both the fruit mesocarp and the seeds of winter squash can be used for consumption, although the focus of breeding efforts varies by cultivar. Cultivars bred for fruit consumption are selected for fruit mesocarp quality traits such as carotenoid content, percent dry matter, and percent soluble solids, while these traits are essentially ignored in oilseed pumpkins. To compare fruit development in these two types of squash, we sequenced the fruit transcriptome of two cultivars bred for different purposes: an acorn squash, ‘Sweet REBA’, and an oilseed pumpkin, ‘Lady Godiva’. Putative metabolic pathways were developed for carotenoid, starch, and sucrose synthesis in winter squash fruit and squash homologs were identified for each of the structural genes in the pathways. Gene expression, especially of known rate-limiting and branch point genes, corresponded with metabolite accumulation both across development and between the two cultivars. Thus, developmental regulation of metabolite genes is an important factor in winter squash fruit quality. Cucurbita pepo is the most genetically and morphologically diverse species within Cucurbita, spanning two subspecies, C. pepo ssp. pepo and C. pepo ssp. texana. Market classes of C. pepo include decorative gourds and pumpkins and oilseed pumpkins, the fruit of which is not consumed, summer squash and zucchini that are eaten at an immature stage, and acorn and delicata winter squash that are consumed when ripe and have a highly refined eating quality. Often in the breeding process, certain characteristics are prioritized at the expense of others in each market class. This contrast is particularly pronounced with oilseed pumpkins, where the fruit is essentially inedible because efforts have focused primarily on improving seed characteristics. These extremes in fruit phenotypes resulting from divergent breeding for fruit or seed consumption provide an excellent opportunity to study the genetic basis of fruit quality in winter squash.C. pepo are β-carotene and lutein.2 Second, starch content is important for winter squash fruit texture; ideally squash have a smooth, slightly dry consistency, which is correlated with higher starch and dry matter content.3 Two negative texture attributes, fibrousness and wateriness, are correlated with low starch and dry matter.3 Third, sugar content is correlated with perceived sweetness of winter squash, with sucrose as the most important predictor.4 Sweetness is thought to be important for consumer acceptability and to influence perception of overall squash flavor, as squash flavor was positively correlated with perceived sweetness in taste panels.5 Percent soluble solids, an efficient trait to phenotype, is also correlated with sucrose levels and perceived sweetness.6 Therefore, by measuring carotenoids, starch and sugar, it is possible to capture many of the important aspects of fruit quality.Fruit quality is one of the most important breeding goals for winter squash. Although fruit quality involves the complex interplay of flavor, texture and appearance, there are three easily measured metabolites that have a major impact on quality. First, carotenoids are the source of winter squash fruit color and provide some of the greatest nutritional benefits of eating squash. The primary carotenoids in 8 Carotenoids also steadily accumulate after 30 DAP and in storage.9These three fruit quality metabolites are synthesized and accumulate in predictable patterns throughout fruit development. The first stage in fruit development is fruit expansion, where the new fruit quickly grows to reach its maximum size 15–24 days after pollination (DAP). At the same time as fruit expansion, starch accumulates in the growing fruit, reaching maximum dry matter around 30 DAP. At this time, sugars start to accumulate, and eventually, especially after harvest, starch is degraded and sugars continue to accumulate.10 pepper,12 grape,13 banana14 and more. Such studies have successfully captured the expression of metabolic genes involved in the biosynthesis of the metabolites of interest. This technique is especially useful in squash (2n=2x=40), because it does not require a completed genome assembly; there is currently no genome assembly available for squash, which is estimated to have a genome size of 500 Mb.15 Previous squash transcriptome studies have included analyses of summer squash (C. pepo) root, leaf and flower tissue,16 pumpkin (C. pepo) leaf and flower tissue,17C. moschata leaf, stem and shoot tissue,18 and acorn squash (C. pepo) fruit and seed tissue.19 No study to date, however, has used transcriptome sequencing to look at differential gene expression throughout winter squash fruit development.Transcriptome sequencing can provide valuable insight into the gene expression underlying developmental changes during fruit development. It has been used to study fruit development and metabolite accumulation in tomato,19 We also sequenced ‘Lady Godiva’, an oilseed pumpkin that was bred for the consumption of its hull-less seeds and whose mesocarp is not intended for consumption and is discarded after seed extraction. Sequencing their fruit transcriptome at five time points allowed us to examine how metabolic processes act throughout mesocarp fruit development to create squash with contrasting fruit quality phenotypes.In this study, we describe comparative transcriptome sequencing throughout fruit development of two squash bred for different end uses at the mature fruit stage. The first cultivar we sequenced was bred for consumption of the fruit mesocarp: ‘Sweet REBA’. ‘Sweet REBA’ is an pureline acorn squash cultivar with a reference fruit and seed transcriptome that we previously generated.C. pepo, which allowed us to make use of the wider array of genetic resources available in this species. In addition, they were bred for contrasting end uses, reflected in our previous observation of their large differences in fruit quality, which suggested they would provide a useful contrast of fruit quality traits throughout fruit development. Both lines were grown at the Homer C. Thompson Vegetable Research Farm  in summer 2012 using standard horticultural practices. Flowers were manually self-pollinated and mesocarp and seed tissue were collected from fruits at 5, 10, 15, 20 and 40 DAP. Mesocarp samples were taken from a longitudinal slice of the fruit, excluding seed, placental and rind tissue. Approximately 40 ml of chopped tissue per sample was flash-frozen in liquid nitrogen, stored at −80 °C, and used for RNA sequencing and carotenoid analysis. Additional longitudinal slices of mesocarp tissue were used for soluble solids and dry matter analysis. Seed tissue was collected and used for RNA sequencing and transcriptome assembly, but was not used further in this study. Three biological replicates were collected for each genotype per time point, each consisting of two fruits from different plants.‘Sweet REBA’ acorn squash and an inbred line derived from the OP oilseed pumpkin ‘Lady Godiva’ were chosen for this transcriptomic analysis of winter squash fruit quality because they are both inbred lines that are grown commercially as cultivars, which makes them representative of squash in the marketplace. They are both within the same species, et al.20 Percent soluble solids (degrees Brix) was determined by freezing samples, allowing them to thaw and analyzing their juice using a digital refractometer. Percent dry matter was calculated by drying mesocarp samples at 60 °C until they reached a constant weight and then dividing the final weight by the starting weight.Fruit quality traits were phenotyped using mesocarp samples from the same fruits used for RNA sequencing. Carotenoids were measured with high-performance liquid chromatography using the protocol described in Van Eck Total RNA was extracted from mesocarp and seed samples using a Qiagen RNeasy Plant Mini Kit . For each biological replicate, equal amounts of total RNA from two fruit were pooled. The Indiana University Genomics Facility prepared barcoded sequencing libraries using the Illumina TruSeq Sample Prep Kit  and samples were sequenced on three lanes of an Illumina HiSeq 2000.19 were combined with the reads sequenced in this study for transcriptome assembly. Reads were cleaned with Fastq-mcf21 with a minimum read length of 50 bp and minimum base quality score of 30. Read quality was assessed with FastQC22 before and after cleaning. Reads from the same genotype were assembled together with Trinity v2.0.623 to result in two assemblies. Corset v1.0324 was used to assign genes into clusters and the longest transcript was selected from each cluster to use as the representative transcript. Contigs from ‘Lady Godiva’ were queried against the ‘Sweet REBA’ assembly with BLAST25 and any contigs not found or with <90% sequence identity were added to the combined assembly. This representative transcriptome was cleaned of contaminating sequences with SeqClean,26 using plant pathogen sequences as a screening database.27Reads from ‘Sweet REBA’ from a previous analysis28 and TAIR1029 protein databases using BLASTx with a cutoff of 1e−20 to assign putative functions to transcripts. Predicted peptides were analyzed using InterProScan30 to identify functional protein domains and assign Gene Ontology (GO) terms.The transcriptome was screened against the SWISS-PROT, TrEMBL31 then using Cuffdiff32 to calculate FPKM (fragments per kilobase of exon per million reads mapped) values and identify significantly differentially expressed genes. Expression differences were considered significant for P values less than the false discovery rate as determined by the Benjamini–Hochberg correction for multiple tests.32 Carotenoid, sugar and starch metabolic gene homologs were identified based on their annotations and using BLAST.25 FPKM values were log transformed using log2+1 and plotted using the R heatmap.2 function in the gplots package.33Gene expression in fruit mesocarp samples from ‘Sweet REBA’ and ‘Lady Godiva’ was analyzed by mapping reads to the representative transcriptome using bowtie2,Q value, (2) expression value greater than 1 FPKM in at least one stage of development and (3) a log2 fold change greater than 2. A Venn diagram was created to determine overlapping sets of filtered, differentially expressed genes between ‘Sweet REBA’ and ‘Lady Godiva’ at the five stages of development.34 To see which filtered genes showed similar expression over time in each accession, K-means clustering was performed using the Gap statistic35 to determine the number of clusters. The R packages cluster36 and factoextra37 were used for clustering. Overrepresented GO terms were investigated for each cluster using a hypergeometric test and the Benjamini and Hochberg False Discovery Rate as implemented in the BiNGO38 plugin for Cytoscape.39The data sets were then filtered using the following criteria: (1) a significant −1 fresh weight by 40 DAP . The resulting reads were combined with ‘Sweet REBA’ reads from a previous analysissemblies , which wProposed pathways for carotenoid, starch and sugar biosynthesis during winter squash fruit development were assembled from the literature and one or more homologs for all of the structural genes involved were identified using annotation data and BLAST. For instances where multiple homologs were identified, the homolog that was the best fit for our proposed biosynthetic pathway and most consistent with the observed phenotypic differences between the two cultivars was selected. Expression of these fruit quality biosynthetic genes was calculated from the fruit mesocarp transcriptome data across five time points in fruit development.40–42 Most of the genes were either more highly expressed in ‘Lady Godiva’ or had approximately equal expression levels in the two cultivars . Another important step in carotenoid synthesis is PSY, which is a rate-limiting step in the carotenoid biosynthetic pathway in marigolds, canola, and tomato.41 Phytoene synthase was significantly more highly expressed in ‘Lady Godiva’, with 4-fold higher expression at 40 DAP, similar to what was observed in summer squash, where phytoene synthase was much more highly expressed in an orange flesh cultivar as compared with a white flesh cultivar53 and phytoene synthase expression was positively correlated with carotenoid content in some tissues and cultivars,61 and in C. maxima, which had a higher carotenoid concentration and also higher phytoene synthase expression than C. moschata.54 Phytoene synthase has also been suggested to be a central enzyme in controlling carotenoid concentration in watermelon.62 Finally, carotenoid breakdown is also an important determinant of carotenoid levels in fruit tissue. CCDs were not generally differentially expressed in the two cultivars, and thus did not likely influence their differential carotenoid concentrations. This is in contrast to some cultivars of summer squash, where levels of carotenoids in the fruit were inversely correlated with CCD expression.63The differential carotenoid gene expression between ‘Lady Godiva’ and ‘Sweet REBA’ was especially pronounced for the genes known in other species to be important determinants of carotenoid synthesis and degradation. DOXP synthase (DXS), which is the first enzyme in the DOXP pathway that produces the precursors for carotenoids, is predicted to be a regulatory step for carotenoid synthesis in tomatoes.64 In our study, the higher expression of lycopene β-cyclase in ‘Sweet REBA’  is consistent with its higher accumulation of β-carotene and the higher expression of lycopene ε-cyclase in ‘Lady Godiva’  is consistent with its higher accumulation of lutein.From lycopene, either both lycopene ε-cyclase (LYC-E) and lycopene β-cyclase (LYC-B) can act to make α-carotene, which is then converted to lutein, or solely lycopene β-cyclase can act to make β-carotene. In maize, natural variation in lycopene ε-cyclase influences the division of the carotenoid flux through the two branches of the pathway.57We observed a relatively high level of expression of β-carotene hydroxylase  and zeaxanthin epoxidase (ZEP), even though zeaxanthin and violaxanthin did not accumulate to appreciable levels in our squash. This is likely explained by the high levels of CCDs in both cultivars, which degrade carotenoids and could have kept the levels of zeaxanthin and violaxanthin low by degrading them quickly. This phenomenon was also described previously in watermelon.47 Alkaline and acid α-galactosidase  both act to break down stachyose and raffinose, and they had high expression levels early in fruit development in our study. Similar patterns of enzyme activity were observed for alkaline and acid α-galactosidase in developing buttercup squash,7 including the higher expression of alkaline α-galactosidase, which they hypothesized was the primary enzyme working to break down stachyose and raffinose.In early fruit development, sugars must be transported to the fruit to provide the precursors for metabolite biosynthesis as well as other developmental processes. The two primary transport sugars in squash are stachyose and raffinose. Sucrose, galactose, glucose, and fructose are also transported.7 In the same study, sucrose synthase activity started high and then declined, but then increased again during the ripening stage. This continued sucrose synthase activity later in fruit development was similar to what we observed. Galactose is broken down by the sequential activity of galactokinase (GK), UDP-glucose/galactose pyrophosphorylase (UGGP), and UDP-glucose epimerase (UGE), which were all expressed at a moderate to high level, especially in the early stages of fruit development. UDP-glucose pyrophosphorylase (UGPase) and PGM were also expressed at a high level, which is in accord with their role in synthesizing glucose-6-P, which is then imported into the amyloplast as the substrate for starch synthesis.Stachyose and raffinose are broken down into sucrose and galactose, which then must be converted to glucose-6-P in order to synthesize starch. Sucrose can be broken down by either acid and neutral invertases (AIN and NIN) or SUS. We observed higher levels of invertase expression in very early fruit development and higher levels of sucrose synthase expression for the remainder of development. In buttercup squash, invertase activity was also high for the beginning of fruit development and then decreased, which was suggested to provide increased osmotic potential early in development for the rapidly expanding fruit.7Overall, the expression of these sugar metabolism genes was generally higher in ‘Sweet REBA’, which likely acted to provide the substrate needed for ‘Sweet REBA’ to synthesize more starch than ‘Lady Godiva’. One exception was alkaline α-galactosidase, which was significantly more highly expressed in ‘Lady Godiva’ and was particularly highly expressed at 10 and 15 DAP. Because ‘Lady Godiva’ was not accumulating as much starch as ‘Sweet REBA’, it may have required higher levels of imported sugars for other developmental processes, such as cellulose synthesis. Sucrose synthase was significantly more highly expressed in ‘Lady Godiva’, and could have also been working to supply substrate for cellulose synthesis, as previously suggested in winter squash.66 and had a significantly higher expression in ‘Sweet REBA’, which could contribute to the greater starch accumulation in ‘Sweet REBA’. Other important regulatory steps in potato starch synthesis are PGM, which converts glucose-6-P imported into the amyloplast into glucose-1-P, and AGPase, which makes ADP-glucose, the first committed reaction in starch synthesis.66 Plastidic phosphoglucomutase had roughly similar expression in the two genotypes, but the large subunit of ADP-glucose pyrophosphorylase had a significantly higher expression in ‘Sweet REBA’. In another study of winter squash, ADP-glucose phosphorylase was thought to be important in determining the level of starch accumulation.56To synthesize starch, glucose-6-P and ATP must be imported to the amyloplast by the glucose phosphate transporter and the amyloplastidial ATP/ADP translocator (AATP) respectively. AATP is a major rate-limiting enzyme for starch synthesis in potatoC. moschata as compared with C. maxima, expression of granule-bound starch synthase was positively correlated with amylose content.56 Differential expression of granule-bound starch synthase and soluble starch synthase varied throughout fruit development, with higher expression in ‘Lady Godiva’ in the middle stages of fruit development, but higher expression in ‘Sweet REBA’ at the end of fruit development. Starch-branching enzyme was significantly more highly expressed in ‘Sweet REBA’ at the final three stages of development. These expression differences may correspond to differential ratios of amylose and amylopectin in these two cultivars.Several different enzymes work together to synthesize starch from ADP-glucose. Granule-bound starch synthase (GBSS) makes amylose, which is the unbranched form of starch. Soluble starch synthase (SS), starch-branching enzyme (SBE), and debranching enzyme (DBE) work together to synthesize amylopectin, the branched form of starch. In 48 A similar developmental transition occurs in kiwifruit, where α-amylase activity increases after peak starch accumulation and soluble solid levels start to rise.67 In banana, which also accumulates starch and converts it to sugar during ripening, α-amylase activity, and then α-glucosidase and β-amylase activity, coincided with the onset of ripening and starch degradation68 and β-amylase activity was correlated with a decrease in starch and an increase in total sugars.69 Alpha-amylase and α-glucosidase were significantly more highly expressed in ‘Sweet REBA’ at one or more time points, providing more substrate for sucrose synthesis.After winter squash fruit reach full size and maximum dry matter, they shift from accumulating starch to degrading starch and accumulating sucrose. During this shift, starch degradation genes increase in expression. We noted a large increase in α-amylase (AMY) expression but a relatively constant level of β-amylase (BAM) expression, similar to enzyme activity patterns observed in buttercup squash.48 We observed high α-amylase expression, but also high expression of SP, which is responsible for phosphorolytic starch breakdown. Starch phosphorylase, however, is also involved in starch synthesis, so because its expression was especially high during early-to-mid fruit development, perhaps it was primarily involved in starch synthesis, leaving hydrolytic breakdown as the predominant starch breakdown pathway in squash.Starch breakdown can be phosphorolytic or hydrolytic. Hydrolytic breakdown via α-amylase and β-amylase was thought to be the primary method in squash due to measurable α-amylase and β-amylase activity and the detection of maltose, a product of hydrolytic breakdown.The products of starch breakdown are exported from the amyloplast and then converted into fructose-6-P and UDP-glucose, the two substrates for sucrose synthesis. The sugar transporters glucose phosphate transporter (GPT) and MEX1 were significantly more highly expressed in ‘Sweet REBA’, indicating that more starch breakdown products may have been leaving the amyloplast. In addition, most of the enzymes involved in the conversion to fructose-6-P and UDP-glucose were significantly upregulated in ‘Sweet REBA’ at one or more time points: α-glucosidase, PGM, UGPase and phosphoglucose isomerase (PGI).70 In melon, sucrose-phosphate synthase activity was correlated with sucrose accumulation between cultivars that accumulated different levels of sucrose.71 The same relationship was seen in watermelon, in which sucrose-phosphate synthase activity was higher in cultivars that accumulated more sucrose72 and was also differentially expressed across fruit development.62 Sucrose-phosphate synthase and sucrose-phosphate phosphatase were both significantly differentially expressed at all five developmental time points sampled, with higher expression in ‘Sweet REBA’.The final steps of sucrose synthesis are the sequential action of sucrose-phosphate synthase (SPS) and sucrose-phosphate phosphatase (SPP) to yield sucrose. The same final steps in starch to-sugar conversion are seen in banana, where sucrose-phosphate synthase activity increases as starch is being degraded and sucrose starts to accumulate. Sucrose-phosphate synthase is a major enzyme that is associated with sugar accumulation.Taken together, the identification of starch and sucrose metabolic genes and analysis of their differential gene expression demonstrates that ‘Sweet REBA’ accumulates more starch because of higher expression of sugar metabolism and starch synthesis genes. This higher level of stored starch, in combination with higher expression of starch breakdown and sucrose synthesis genes, then permits ‘Sweet REBA’ to accumulate more sucrose while maintaining a high level of starch.8 which are less likely to vary between genotypes. The 40 DAP time point, in contrast, is after fruit expansion has completed and around the time fruits reach maximum dry matter.8 As the total dry matter content has a major effect on later fruit quality, this time point is when we would expect to see the gene expression differences responsible for fruit quality differences. In addition, the over-representation of differentially expressed fruit quality-related unigenes at 40 DAP is consistent with the phenotypic data in this study, which showed the highest contrast in fruit quality traits between the two genotypes at 40 DAP.Towards a global view of gene expression in developing winter squash fruits, we identified unigenes that were differentially expressed between ‘Sweet REBA’ and ‘Lady Godiva’ at each time point and determined which GO terms were enriched in these data sets. No GO terms directly related to fruit quality were enriched in any of the data sets except for 40 DAP. This is consistent with our knowledge about squash fruit development, because the first four time points are during the initial fruit expansion and accumulation of dry matter stages,The specific fruit quality-related GO terms overrepresented at 40 DAP are also consistent with our observed phenotypes. The unigenes upregulated in ‘Sweet REBA’ are enriched for GO terms related to carbohydrate metabolism, including hexose, disaccharide, oligosaccharide, and polysaccharide metabolic processes. This pattern matches the higher expression in ‘Sweet REBA’ of the starch and sucrose synthesis genes described earlier, as well as ‘Sweet REBA’s greater accumulation of starch and sucrose as compared with ‘Lady Godiva’. The unigenes upregulated in ‘Lady Godiva’ are enriched for the ‘pigment metabolic process’ GO term, which is expected based on ‘Lady Godiva’s greater accumulation of carotenoid pigments and the higher expression of important carotenoid biosynthetic genes in ‘Lady Godiva’.After identifying unigenes differentially expressed across fruit development in either ‘Sweet REBA’ or ‘Lady Godiva’, we clustered the identified unigenes by expression pattern and looked at the GO term enrichment in each cluster. Overall, there was a pattern to the types of GO terms enriched in these unigenes. Some of the most commonly represented GO term categories were carbohydrate metabolism, cell wall development, and regulation of metabolic processes. This is in accord with the major processes occurring in fruit development: fruit growth and expansion and the accumulation of carbohydrate and other metabolites.8 and thus active cell wall biosynthesis. ‘Sweet REBA’ cluster 1 and ‘Lady Godiva’ cluster 4 had a steady, higher than-average expression across fruit development and were enriched for carbohydrate metabolism-related GO terms. Some of the starch metabolism genes highlighted above were also members of one or both of these clusters. This above-average expression was consistent with carbohydrate’s role as the primary type of storage molecule in winter squash fruit.Generally, the GO term enrichment corresponded logically with the individual cluster expression patterns. ‘Sweet REBA’ cluster 6 and ‘Lady Godiva’ cluster 6 had expression patterns that decreased across fruit development and were enriched for GO terms related to negative regulation of metabolic processes. The decrease in negative regulation over time could explain the observed higher expression of some of the fruit quality biosynthetic genes towards the end of fruit development, as well as the higher accumulation of the fruit quality metabolites at the later time points. ‘Sweet REBA’ cluster 7 and ‘Lady Godiva’ cluster 1 were enriched for cell wall and carbohydrate metabolism-related GO terms. Although they maintained a fairly average expression across fruit development, they peaked slightly from 10–20 DAP, especially in ‘Lady Godiva’, which is the period of major fruit growthC. pepo and related species. Furthermore, this resource will be useful for molecular-informed breeding of oilseed pumpkins and squash with an edible mesocarp toward a goal of reducing food waste while maintaining oilseed quality and yield.In this study, we sequenced the fruit transcriptome of an acorn squash, ‘Sweet REBA’, and an oilseed pumpkin, ‘Lady Godiva’, across fruit development to explore the key gene expression differences that underlie the contrasting metabolic processes that affect winter squash fruit quality. These two cultivars were developed for different purposes, fruit versus seed consumption, and accordingly had a resulting contrast in the important fruit quality traits of carotenoid content, percent dry matter, and percent soluble solids. Putative metabolic pathways for the synthesis of carotenoids, starch and sugar were assembled from the literature and expression of the structural genes of these pathways varied both across fruit development and between the two cultivars. The overall trend was increased expression of metabolic genes during times of metabolite accumulation and in the cultivar that accumulated higher metabolite levels. This reveals the transcriptional regulation of metabolic gene expression that may be an important determinant of fruit quality. It will be important to investigate the expression patterns of these genes in additional winter squash cultivars in future studies to determine the degree to which these gene expression profiles describe metabolisms that lead to improved fruit quality in winter squash in


Each human genome is a blueprint, sculpted by millennia of natural selection, and individualised through meiosis. Its decoding will enable formulated health-related decisions that will become an increasing part of our daily existence.I believe that we will look back upon 2015 as the year when whole-genome sequencing (WGS) went mainstream, and the concept of npj Genomic Medicine is a new international, peer-reviewed journal dedicated to publishing the most important scientific advances in genomics as applied to the practice of medicine. As its inaugural editor, my working definition of genomic medicine is as follows: diagnosis, prediction, prognosis, prevention and/or treatment of disease and disorders of the mind and body, using approaches informed or enabled by knowledge of the genome and the molecules it encodes.1 Our goal is to publish outstanding papers that describe genome-related studies of individuals, families or populations in a medical context. We will emphasise the coupling of detailed phenotype and genome sequence, genotype, or mutational information in delineating the underlying aetiology of disease.n=1. I can see a time in the near future where such studies of individuals carrying presumed highly penetrant mutations but who are ‘resilient’ for that particular disorder will rival the number of papers describing patients who do succumb to their genetic liability. We will also encourage sharing of ideas, as well as recommendations and/or guidelines of how such data should be used in the clinical management of study subjects and others.The journal will also welcome detailed case or family reports where 2–7 (2) WGS applied to non-invasive prenatal diagnosis;9 (3) WGS characterisation of the ~99% of the genome not readily captured through less comprehensive technologies11, (4) discovery of new regulatory variants related to risk of disease;13 (5) new approaches including computational algorithms, population genetics and/or functional experimentation for interpreting data;14–20 (6) databases to allow universal access of massive genomic data sets for research;21 and (7) all of the above integrated and interfaced with ‘smart phone’ technology and other ‘big data’ innovation, which is part of our inevitable future.Crystal gazing, based on my own experiences and those of our distinguished Associate Editors, I foresee that in the journal’s first few years, the most impactful areas we hope to publish on will include the following : (1) WGS in clinical diagnosis and prognosis;22–25 In my own research area of autism genomics, I am increasingly enthralled how cultural evolution driven by the information era may be influencing our natural evolution, and what the consequences are for medicine in the twenty-first century26.As genomic information may inform on past, present and future outcomes of a personal and sensitive nature, it warrants handling with thought and care. We will encourage papers in areas of ethics, health economics and law, as they apply to medical genomics and influence our rapidly changing society.Nature, the material published in npj Genomic Medicine will also be disseminated broadly via social media.To capture all of these advances, the journal will publish Articles, Case or Resource Reports, Brief Communications, Commentaries and Review Articles. A professionally written Editorial Summary will accompany each Article, summarising the key issues addressed. The speed of moving papers through the peer-review system will be paramount to our success, but only those papers deemed outstanding and strategic to the journal’s mission will be published. With extensive support from Nature guidelines to ensure these standards are upheld. The way these often large and complex data sets are handled, stored, shared and analysed is evolving, falling into the realm of what is now called ‘big science’, something the journal will also cover.The journal recognises the importance of the deposition of published data in the appropriate accessible databases and it will follow existing medium26 to deliver the message of science to continue to build a global village striving for the optimal health of all of its inhabitants.The impact of genomics is now ubiquitous in health research, both basic and clinical, and must be supported by impactful worldwide journal representation. We will wholeheartedly strive to have as many members from as many different countries and backgrounds involved in the different aspects of journal. Diversity drives our genetic history and we believe it is also crucial to the journal’s success. In fact, in agreeing to accept the role of editor, one of my main motivations was to try to use genomic medicine as the