The proposed algorithm computes the Normalized Average Absolute Difference (NAAD) for each segment and uses it as a measure to determine if the segment has sufficient colour variations. The initial colour constancy adjustment factors for each segment with sufficient colour variation is calculated. The Colour Constancy Adjustment Weighting Factors (CCAWF) for each pixel of the image are determined by fusing the CCAWFs of the segments, weighted by their normalized Euclidian distance of the pixel from the center of the segments. Results show that the proposed method outperforms the statistical techniques and its images exhibit significantly higher subjective quality to those of the learning-based methods. In addition, the execution time of the proposed algorithm is comparable to statistical-based techniques and is much lower than those of the state-of-the-art learning-based methods.Digital camera sensors are designed to record all incident light from a captured scene, but they are unable to distinguish between the colour of the light source and the true colour of objects. The resulting captured image exhibits a colour cast toward the colour of light source. This paper presents a colour constancy algorithm for images of scenes lit by non-uniform light sources. The proposed algorithm uses a histogram-based algorithm to determine the number of colour regions. It then applies the K-means Image sensors built into today’s digital cameras mostly use either the Charge Coupled Device (CCD) or Complementary Metal-Oxide Semiconductor (CMOS) technology. Both CCD and CMOS are semiconductor devices that serve as “electronic eyes”. While they both use photodiodes, they differ in terms of manufacturing. A CCD sensor is an array of capacitors, each of which can store its own electrical charge. Groups of capacitors on the CCD form pixels, which are charged using the photoelectric effect. This happens when the capacitor converts an incident photon of light into an electrical charge. When a circuit is connected to the pixels, the value of the charge is then numerically measured and recorded in a computer file. After recording, the image can be displayed. Most CCD sensors use a Bayer filtration pattern for their pixels; each pixel is covered with either a red, blue, or green filter, which only allow light that is that colour to reach the capacitor. In this filtration pattern, there are twice as many green sensors as red or blue, because human eyes are more sensitive to green than to other colours. Since it is known which sensor has which colour filter, the intensity of red, green, and blue light can be determined anywhere on the CCD sensor . The forCMOS sensors are much cheaper to produce than CCD sensors, as they are made of the same semiconductor fabrication lines used in microprocessor and static RAM memory chips. Consequently, they have largely replaced CCD sensors, which rely on more specialised fabrication methods. Each photosensitive pixel within the CMOS sensor is comprised of a photodiode and three transistors. One transistor is responsible for activating and resetting the pixel, the second amplifies and converts the stored charge within the photodiode to voltage and the third performs selection and multiplexing. The signals within each pixel are multiplexed by row and column to multiple on chip analog-to-digital converters, resulting in a high-speed yet low-sensitivity image capture system. Furthermore, CMOS sensors are susceptible to high fixed-pattern noise due to manufacturing flaws in the charge to voltage conversion circuits. As a result of their multiplexing design, CMOS sensors are often paired with electronic rolling shutters, although versions with additional transistors can be used in conjunction with global shutters and simultaneous exposure. The low sensitivity of a CMOS sensor results in a lower power consumption and the ability to handle higher light levels than a CCD sensor, resulting in their use in special high dynamic range cameras . CCD andFurthermore, digital camera sensors are designed to record all incident light from the scene but they are unable to distinguish between the colour of the light source and the true colour of objects. This results in the captured image exhibiting a colour cast, representing the colour of the light source ,6,7,8,9.Researchers have proposed various colour constancy adjustment methods to address the problem of colour constancy in the presence of both single and multiple light sources in digital images ,43,44,45c is the image colour component and n is the order of the derivative, p is the Minkowski-norm, and The Grey World , the Maxp. When p = 1, Equation (1) becomes the Grey World method, which assumes the average value of all colour components within an image are achromatic. If p = ∞, Equation (1) represents the Max-RGB colour constancy algorithm, which assumes that the maximum values of the image colour components are achromatic. By setting p to be equal to 6, Equation (1) becomes the Shades of Gray algorithm, which is less data dependent than both the Gray World and White Patch colour correction methods. Equation (1) can also represent higher order colour constancy methods, including Grey Edge-1 and Grey Edge-2 by setting p = 1, n = 1 or 2, respectively. These two approaches assume that derivatives of the image colour components are achromatic. The Weighted Grey Edge method was proposed by Gisenji et al. in [Equation (1) can be used to represent different statistical colour constancy algorithms by using various values of t al. in . This cot al. in .Forsyth  introducSeveral learning-based colour constancy adjustment methods have recently been reported in the literature. The colour constancy problem as a 2D spatial localization task in a log-chrominance space was formulated by Baron in . Baron oTo perform colour constancy for images of scenes illuminated by multiple light sources, researchers have proposed techniques which estimate the local incident light based on the assumption that the colour of the incident light of a mini region is uniform ,31,32,33A biologically inspired model for colour constancy was reported by Gao et al.  that expg et al. . The autg et al.  proposedYang et al.  proposed++ clustering method [The aforementioned algorithms work reasonably well when the scene is illuminated by a uniform light source and lacks large uniform colour areas within the image, but the performance of existing algorithms deteriorates in the presence of large uniform colour areas and when the scene is illuminated by multiple non-uniform light sources. This paper presents a colour constancy algorithm for images of scenes lit by multiple non-uniform light sources (this paper is an extended version of a previous paper ). The prg method  is then The proposed Colour Constancy Adjustment by Fusion of Image Segments’ initial colour correction factors (CCAFIS) algorithm is divided into three steps: automatic image segmentation, segment selection and calculation of initial colour constancy weighting factors for each segment and calculation of the colour adjustment factors for each pixel. Each of these three steps is detailed in the following sub-sections.th of the total number of image pixels, respectively. These numbers were empirically found to be effective in finding the reasonable number of segments when dealing with images of five different image datasets. The calculated number of segments and the ++ clustering algorithm, which divides the input image pixels into several segments based on their colour properties.The proposed automatic image segmentation algorithm first converts the input RGB image into a grey image. It then splits the coefficients within the resulting grey image into a histogram with 256 bins. The resulting histogram is then filtered using the following Gaussian low-pass filter: In this section, each of the resulting segments are processed independently to circumvent the segments containing uniform areas and to calculate initial colour adjustment factors for each segment, as follows:The proposed technique first calculates the Normalized Average Absolute Difference (NAAD) of pixels for each colour component of the segment using Equation (2):The resulting lgorithm , as writIn this research, the Grey World colour constancy method, which is one of the most effective and yet computationally inexpensive techniques compared to other statistical colour constancy algorithms ,3, is usd1, d2, …, and dn are the Euclidian distance of the pixel i from the centroid of the segments 1, 2,.., n, respectively.In this section, the fusion of the initially calculated colour constancy adjustment factors for the selected segments to calculate per pixel colour correction weighting factors is discussed. The algorithm is then fed the calculated initial colour constancy adjustment factors of the selected segments, the gravitational centroid of each selected segment’s pixels and the Decision Vector (DV). The proposed algorithm calculates the Euclidian distance of each pixel from the centers of the selected segments and uses them to regulate initially calculated colour adjustment factors to determine per pixel weighting factors using Equation (4):This balances the effect of colours of different light sources on the colour of each pixel. The resulting weighting factors are used to colour balance the input image using The Von-Kries Diagonal model , as showIn this section, the performance of the proposed Colour Constancy Adjustment by Fusion of Image Segments’ initial colour correction factors (CCAFIS) is assessed on five benchmark image datasets, namely: Multiple Light Source (MLS) , The MulThe performance of the colour constancy algorithms are generally assessed both objectively and subjectively. The angular error, also known as the recovery angular error, is an objective criterion that is widely used to assess the colour constancy of the images which measures the distance between the colour-corrected image and its ground truth . A lowerRecently, Finlayson et al.  have criBoth recovery and reproduction angular error have been used to assess the objective quality of the colour-corrected image by computing the average of the mean or median recovery/reproduction angular errors of different methods on a large set of colour-corrected images and using them for comparison purpose. The images of the method that have the lowest average of the mean or median recovery/reproduction angular errors have the highest colour constancy.Because human eyes are the final judge for assessing the colour constancy of images, a subjective evaluation is generally considered to be the most reliable assessment method when evaluating colour constancy algorithms. Mean Opinion Score (MOS) is a popular subjective evaluation method that is widely used to compare the visual quality of the images. To determine the MOS for images subjected to different colour constancy adjustment methods, a set of images of different scenes containing diverse backgrounds, foregrounds, objects and a range of colour variations that are taken under various lighting conditions is first chosen. These images are then colour-corrected using different colour balancing techniques. The resulting images are shown to observers who score the images based on their colour constancy . The MOS of each method finally calculated by computing the average score of its images.Objective criteria are widely used to assess the performance of different colour constancy techniques due to their simplicity. However, there is significant debate on the merit of objective measurements and their relation to subjective assessment. Some researchers have argued that objective measurements may not always be in agreement with the subjective quality of the image ,54. ConsTo visualize the effectiveness of the proposed method in dividing the image into several segments and identifying segments with uniform colour areas, a sample image from the UPenn images dataset  is takenR, TG and TB components can efficiently eliminate segments with uniform areas [The Normalized Average Absolute Differences (NAAD) of the three colour components of the segments of the image shown in rm areas . Hence, In the next two sub-sections, subjective and objective results for the proposed method will be presented.To demonstrate the subjective performance of the proposed Colour Constancy Adjustment by Fusion of Image Segments’ initial colour correction factors (CCAFIS) method and to compare the quality of its colour-corrected images with those of the state of the art colour constancy techniques, two sample images from the Colour Checker and the Upenn Natural Image benchmark image datasets are selected and colour-balanced using different colour correction methods. To give the reader a better understanding in the performance of the proposed CCAFIS method on images of scenes with spatially varying illuminant distribution, an image from the MLS image dataset  that repTo generate the Mean Opinion Score (MOS) for the images of the proposed and the state-of-the-art colour constancy methods, a set of images from the Grey Ball, the Colour Checker and the MIMO image datasets, which contain images of scenes lit by either single or multiple light sources, was chosen. The selected images were colour-corrected using the proposed CCAFIS method, as well as other state-of-the art techniques including the multiple illuminant methods such as: Gijsenij et al. , MIRF 33 and CheTo evaluate the objective performance of the proposed method, Grey world , Max-RGBFrom According to The average mean and median recovery angular errors of the colour-balanced images from the MIMO image dataset for different techniques were computed and tabulated in From From ® Core (TM) i3-6006U CPU with a 1.99 GHz processor, 4.00 GB of RAM, and without any additional dedicated graphic processing unit. These methods were timed when applied to colour balance the first 100 images of the Colour Checker benchmark image dataset. For the learning-based techniques, the second 100 images from the Colour Checker benchmark image dataset were used for training. The cumulative execution time for testing the proposed CCAFIS, statistical-based and learning-based methods were measured and tabulated in To enable the reader to compare the execution time of the proposed Colour Constancy Adjustment by Fusion of Image Segments’ initial colour correction factors (CCAFIS) algorithm with both statistical-based and learning-based state of the art colour constancy techniques, the proposed method, Gray World, Max-RGB (White Patch), Gray Edge-1, Gray Edge-2, Exemplar-based , Gray Pi++ clustering algorithm along with a histogram-based method to divide the input image into several segments with similar colour variations. The Normalized Average Absolute Difference (NAAD) of the resulting segments are then calculated and used as a measure to identify segments with uniform colour areas. These segments are then excluded from the calculation of colour constancy adjustment factors for the whole image. The initial colour constancy-weighting factor for each of the remaining segments is then calculated using the Grey World method. The colour constancy adjustment factors for each pixel is finally computed by fusing the initial colour constancy of the remaining segments, regulated by the Euclidian distances of the pixel from the centroids of all remaining segments. Experimental results on both single and multiple illuminant benchmark image datasets showed that the proposed method gives a significantly higher performance to those of the state-of-the-art statistical-based techniques. Furthermore, the proposed techniques gave higher or very competitive results to those of learning-based techniques at a fraction of the computational cost.This paper presented a colour constancy algorithm for non-uniformly lit scenes’ images. The algorithm uses the K-means


Case Report. A 40-year-old male with posttraumatic nonunion patella with quadriceps contracture since 6 months presented with complaints of defect in the left knee with restriction of movements. X-ray of the left knee confirmed our findings. He underwent quadricepsplasty with double-tension band wiring for the patella followed by rigorous physiotherapy to achieve the current level of the knee flexion of 110 degrees.  Nonunion patella with quadriceps contracture is an unusual orthopaedic finding. Very few cases have been recorded in the past with this complication. We present a case of a 40-year-old male with nonunion patella with quadriceps contracture secondary to trauma We conclude that quadricepsplasty with tension band wiring and neutralization wire is one of the good modalities of treatment for a nonunion patella associated with quadriceps contracture. Fractures of the patella contributes to 1% of all skeletal injuries . The thiA 40-year-old male presented with complaints of instability and defect in his left knee since 6 months. The patient gave a history of trauma 6 months back and did not take any treatment for this. Clinically, anterior defect was present over the left knee with visibility of intercondylar articulating surfaces of the tibia and femur. Swelling was seen in the anterior aspect of the left distal third thigh, which, on palpation, was the superior part of the patella. The lower pole of the patella was palpable just above the left tibial tuberosity . The X-rThe patient underwent surgery by anterior approach where quadricepsplasty and tension band wiring for the patella were performed after bringing the superior fragment down . AnotherDuring the immediate postoperative period, the patient was started on dynamic quadriceps strengthening and active straight-leg-raising exercises. After suture removal, continuous passive motion for his knee was added. On discharge, the range of knee motion was from 5 degrees of extension lag to 40 degrees of flexion. At 6 weeks of postoperative follow-up, the patient had a 5- to 90-degree knee motion. The range of motion improved to 0–110 degrees at 3 months follow-up .The aim of reporting this case is to highlight the double-tension wire management and use of single-stage double-tension wire management of an old neglected nonunion patellar fracture. In the procedure, the proximal fragment was mobilized and fixed with the lower fragment using V-Y plasty and double-tension band wires. The objective during the surgery was to achieve fractional lengthening in order to prevent quadriceps lag in patients of nonunion of patellar fractures with large gaps between the fracture fragments. The rate of nonunion in patellar fractures is about 2.7% . There aThe problem with double-stage surgeries is that the presence of Ilizarov or skeletal traction poses a mental trauma to the patient along with surgical complications like bone weakening, pin loosening, pin tract infection, and prolonged duration of treatment. Yet double-stage surgeries have been reported with good results as shown by Dhar and Mir .We opted for single-stage procedure, i.e., proximal fragment mobilization and fixation with the lower fragment/patellar tendon using V-Y/Z-plasty and achieving fractional lengthening. The supplementary fixation with neutralization wire in the region of the patellar tendon not only acted as an internal bracing during the initial rehabilitative period but also as a compressive force that shortens an already contracted patellar tendon. In addition, it also helps in securing the reduction by decreasing the tensile forces, which are exerted by the quadriceps muscle during knee flexion. One major risk with this procedure is overtightening of the neutralization wire resulting in the patella baja. In order to ensure the correct length of the neutralization, an image intensifier should be used intraoperatively to assess and compare the distance of the tibial tuberosity and the distal pole of the patella of the contralateral knee. Moreover, biomechanical assessment of the tension band wire shows that it fails at maximum load of about 695 N after osteosynthesis of the patella. Other methods of treatment like conservative technique and patellectomy are also practiced. In the conservative method, the stability and alignment of fracture fragments are confirmed at 60 degrees of flexion under an image intensifier with no dislocation. The initial restriction of knee flexion is provided by a suitable orthosis, which is gradually increased during the course of treatment. The major risk of conservative management is loss of full extension and stiffness of the knee, which are undesirable especially in a young patient. Patellectomy is another commonly practiced salvage procedure , 8. ThisWe conclude that quadricepsplasty with double-tension band wire management is a good surgical measure in treatment of nonunion patella with quadriceps contracture.


Microbiomes inhabiting plants are crucial for plant productivity and well-being. A plethora of interactions between roots, microbiomes, and soil shapes the self-organization of the microbial community associated with the root system. The rhizosphere  and endosphere  are critical interfaces for the exchange of resources between roots and the soil environment. In recent years, next-generation sequencing technologies have enabled systemic studies of root-associated microbiomes in the endosphere and interactions between roots and microbes at the root-soil interfaces. Genetic factors such as species and genotype of host plants are the driving force of microbial community differentiation and composition. In this mini-review, we will survey the role of these factors on plant–microbe interactions by highlighting the results of next-generation genomic and transcriptomic studies in the rhizosphere and endosphere of land plants. Moreover, environmental factors such as geography and soil type shape the microbiome. Relationships between the root-associated microbiome, architectural variations and functional switches within the root system determine the health and fitness of the whole plant system. A detailed understanding of plant–microbe interactions is of fundamental agricultural importance and significance for crop improvement by plant breeding. In vascular plants, the root system is the main interface for interactions with the soil microbiome. Roots influence biological activity and diversity of microbes in the soil by secreting organic substances including amino acids, carbohydrates and organic acids as well as by depositing root cap border cells and polysaccharide mucilage to soil environments . Thus, rRecent studies have applied high-throughput DNA sequencing to study the composition and organization of different plant microbiomes (16S rRNA gene based community profiling), including both dicot  and monoThe rhizosphere is the narrow soil zone directly surrounding the root system, which is highly modulated by the root system . Roots rHordeum vulgare) shows a distinct microbiome compared to its wild progenitor with respect to function (Human selection during domestication has significant reduced the genetic diversity of modern crop species in comparison to their progenitors . Recent function . Some offunction . Moreovefunction . Neverthfunction . In vegefunction . Similarfunction . Interesfunction . These rAlphaproteobacteria and Ascomycota has been shown in hybrid cultivars in comparison to conventional cultivars (High-yielding maize hybrids display a significantly higher bacterial abundance and more elite probiotic rhizospheric strains with higher productivity of 2,4-diacetylphloroglucinol and superior root-colonization ability in the rhizosphere than their parental inbred lines . It has ultivars . These rThe endosphere, which comprises all inner root tissues is inhabited by microbes . FunctioIt has been shown that beneficial association between host and plant growth promoting rhizobacteria is an inherited trait in the endosphere of tomato plants . MoreoveIn contrast to the model plant Arabidopsis and most dicot plant species, cereal plants display distinct root types including seminal roots and postembryonic shoot-borne crown and brace roots, which show diverse anatomical structures and transcriptomic signatures during development . MoreoveMicrobial diversity of the rhizosphere and endosphere is regulated by physical and chemical characteristics of the rhizosphere. These are partly determined by the species and genotype of the host root. Nevertheless, it is still unclear whether the phylogenetic distance between genotypes is correlated with the microbial community composition in the rhizosphere . DiffereBoth PY and FH contributed to the writing of this mini review.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Rabies is a neglected zoonotic disease with veterinary and public health significance, particularly in Africa and Asia. The current knowledge of the epidemiology of rabies in Mozambique is limited because of inadequate sample submission, constrained diagnostic capabilities and a lack of molecular epidemiological research. We wanted to consider the direct, rapid immunohistochemical test (DRIT) as an alternative to the direct fluorescent antibody (DFA) for rabies diagnosis at the diagnostic laboratory of the Central Veterinary Laboratory (CVL), Directorate of Animal Science, Maputo, Mozambique. Towards this aim, as a training exercise at the World Organisation for Animal Health (OIE) Rabies Reference Laboratory in South Africa, we performed the DRIT on 29 rabies samples from across Mozambique. With the use of the DRIT, we found 15 of the 29 samples (52%) to be negative. The DRIT-negative samples were retested by DFA at the OIE Rabies Reference Laboratory, as well as with an established real-time Polymerase chain reaction, confirming the DRIT-negative results. The DRIT-positive results (14/29) were retested with the DFA and subsequently amplified, sequenced and subjected to phylogenetic analyses, confirming the presence of rabies RNA. Molecular epidemiological analyses that included viruses from neighbouring countries suggested that rabies cycles within Mozambique might be implicated in multiple instances of cross-border transmission. In this regard, our study has provided new insights that should be helpful in informing the next steps required to better diagnose, control and hopefully eliminate rabies in Mozambique. Lyssavirus genus and accounts for tens of thousands of human deaths every year  (WHO Canis mesomelas) and the bat-eared fox  are also able to maintain cycles of rabies and subsequently contribute to the spread of the disease , is a member of the ris) WHO . SeveralThe first introduction of rabies into the KZN province of South Africa probably occurred in 1961 when the disease spread from the Maputo district in southern Mozambique to the northern regions of the KZN province Mansvelt . The higThe classical method for rabies diagnosis is the DFA test , which relies on detecting the presence of lyssavirus antigens by means of a fluorescein isothiocyanate-labelled antibody , rabies diagnostics in Mozambique have been inconsistent because of poor surveillance capacity, with specific reference to limited sample submissions and lack of diagnostic capabilities. As an illustration of the consequence of such limited surveillance, the number of human rabies cases  for some years outnumbered confirmed rabies cases in the reservoir and vectors species, With a view to simplify and improve the rabies diagnostic capability at the CVL in Mozambique, we implemented the DRIT in a training exercise using a cohort of 29 samples previously confirmed as DFA-positive in Mozambique. We also sequenced the G-L RABV cDNA obtained from positive samples towards obtaining a molecular epidemiological view of the relationships among these viruses and with those viruses known from countries sharing borders with Mozambique. In this study, we aim to determine whether active cross-border spread of rabies between Mozambique and its neighbouring countries occurs, as this finding is important in structuring and evaluating regional plans for rabies control and elimination.This study was carried out at the OIE Rabies Reference Laboratory at the Agricultural Research Council-Onderstepoort Veterinary Institute (ARC-OVI), Rabies Division, South Africa. A total of 29 brain tissue samples  were subjected to routine DFA testing at the CVL in Maputo, Mozambique and all of the samples were diagnosed rabies-positive. The samples formed part of a repository of samples stored below -20 °C for an extended period of time (1993–2013) at the CVL . The samn = 29) were subjected to the direct, rapid immunohistochemical test (DRIT) diagnostic assay according to the standard operating procedure using a biotinylated PAb preparation   for 10 min. After fixation, the touch impressions were rehydrated by dip rinsing the slides in TPBS buffer  and then submerged in 3% hydrogen peroxide (Merck) for 10 min at room temperature in order to halt all endogenous peroxidase activity. Subsequent to the hydrogen peroxide flooding, the slides were dip rinsed in fresh TPBS buffer and the excess buffer was shaken from the slides. The areas surrounding the smear impressions were blotted dry using fresh paper towel. Biotinylated PAb preparation (1:600 working concentration) was applied dropwise until the impression was completely covered. After the application of the antibody, the slides were placed in a humidity chamber, incubated at room temperature for 10 min and subsequently dip rinsed in fresh TPBS buffer. The excess buffer was shaken from the slides, and the areas surrounding the smear impressions were blotted dry using a fresh paper towel. All of the touch impressions were covered in a ready-to-use solution of 2 μg/mL streptavidin-peroxidase , after which the slides were transferred to a humidity chamber. The humidity chamber was incubated at room temperature for 10 min and the slides were dip rinsed in fresh TPBS buffer. The excess buffer was shaken from the slides and the areas surrounding the smear impressions were blotted dry using a fresh paper towel. The impressions were covered in a working solution of the 3-amino-9-ethylcarbazole (AEC) chromogen (Sigma-Aldrich), and the slides were transferred to a humidity chamber and incubated at room temperature for 5 min. After sufficient staining had occurred, the slides were submerged in distilled water. The touch impressions were counterstained with a 1:2 dilution of Gill’s formulation #2 (Sigma-Aldrich) for 2 min before they were dip rinsed in distilled water in order to wash away the residual counterstain. Finally, the slides were mounted with a water-soluble mounting medium (1xPBS [Whitehead Scientific]/glycerol [Sigma-Aldrich] prepared 1:1) and examined by light microscopy  at both 200x and 400x magnification in order to score the respective immunoreactivity based on both the presence and staining intensity of the visible red inclusions present on the blue cellular background . One technician from the ARC-OVI Rabies Division performed the DFA test, and two experienced microscopists interpreted the results in a blind reading in order to eliminate bias.The DFA diagnostic assay CDC  was repen = 29) was extracted using the TRI Reagent® (Sigma-Aldrich) according to the manufacturer’s instructions. A reverse transcription polymerase chain reaction (PCR) was performed on the 14 confirmed rabies-positive samples using the G(+) and L(-) primers .The total RNA of all of the neuronal tissue samples  and sequenced using an ABI 3100 automated capillary sequencer situated at the University of Pretoria. With the use of the CLC Main Workbench software , the consensus sequences were trimmed to 592 nucleotides (nt), representing the cytoplasmic domain of the glycoprotein gene and the adjacent G-L intergenic region of the RABV genome . The final sequences were subsequently submitted to the National Center for Biotechnology Information (NCBI) GenBank and allocated unique accession numbers (KM262037-KM262049) (Both the forward and reverse strands of the purified PCR amplicons were sequenced using the PCR primers and the BigDyeM262049) .All of the negative samples from the DFA at the ARC-OVI, Rabies Division were subjected to an established ‘one-step’ quantification real-time PCR assay  . The rooAn alignment of the collection of sequences was created using the ClustalW subroutine of the BioEdit software Hall  and a maFourteen of the 29 samples (48%) were found to be lyssavirus-positive using the DRIT. All of the samples from Mozambique were retested using the DFA at the ARC-OVI laboratory in South Africa in order to confirm the DRIT results. The results from the DFA in South Africa correlated with those of the DRIT . These fn = 14 rabies-positive by DRIT and DFA and n = 15 rabies-negative by DRIT and DFA), were subsequently confirmed by PCR , Swaziland and South Africa  . Our resThe second clade, Clade B, consisted of RABV sequences from RABV-infected dogs found in Mozambique (Manica and Nampula provinces), Zimbabwe (Masvingo province) and South Africa (Limpopo province) . EvidencThe third clade, Clade C, highlighted genetic relatedness between isolates originating from Mozambique (Cabo Delgado and Nampula provinces), Malawi and Zimbabwe  . To our We analysed samples that were found to be rabies-positive and were subsequently stored at -20 °C at the CVL in Maputo, Mozambique. After shipment to our laboratories in South Africa, 48% of these samples could be confirmed as rabies-positive with the use of DFA, DRIT and real-time PCR. There could be several reasons for the negative result in a fairly high percentage of the samples. Viral tropism could be one such reason, as only a portion of the brain material was shipped. However, all of the samples were homogenised prior to testing, which limits the likelihood of poor antigen distribution. A more likely possibility could be the degradation of antigens and genetic material to the point where these could not be detected by any of the methods we have applied. The maintenance of constant temperature storage of brain tissue samples subsequent to the application and interpretation of any diagnostic test is of the utmost importance  in 2009. The KZN province was selected as the South African study location because of the unique challenges the province faces for rabies control and elimination. KZN has achieved large-scale success since the programme was initiated, and reached a milestone in 2010 when the province celebrated 1 year without a diagnosed case of human rabies for the first time in 20 years  .It is important to note that the large empty geographical spaces separating the isolates clustering in Clade C  and 3 doThe only laboratory capable of rabies diagnosis (DFA test) in Mozambique is situated in the capital of Maputo (Maputo province), which is located at the southern tip of the country. Poor roads and the long distance samples have to travel from the north of the country to the CVL could explain why so few samples originate from the northern provinces of the country. To improve sample submission in a cost-effective way, the decentralisation of rabies diagnosis would decrease the distance samples have to be transported, whilst theoretically increasing the number of samples submitted. Because of the low capital investment requirements and comparable diagnostic sensitivity, the DRIT would be the ideal test for decentralised implementation and could serve to provide a more accurate representation of the rabies burden in the surrounding area. Through the use of the DRIT, surveillance can be improved in areas where it has typically been lacking.It has been demonstrated on various occasions that the only feasible way to eliminate human rabies is to control and eliminate the disease from canine populations. The strategic vaccination of dog populations should be directed by effective, accurate and cost-effective surveillance networks, as illustrated in this study through the use of the DRIT. Although the establishment of a foundational surveillance system is of utmost importance, it is clear that basic epidemiological information is not sufficient if a higher epidemiological resolution is needed. Basic burden data coupled with molecular epidemiology can improve the resolution of surveillance by determining focal points for rabies intervention.In support of previous observations, this study further demonstrates the cross-border spread of rabies between neighbouring countries in Africa, thus impacting disease control and elimination efforts for the region as a whole. Elimination of canine rabies is possible and should be the ultimate objective of rabies control initiatives. To achieve this, regional and continental strategies that rely on and encourage effective national programmes are needed. Without the cooperation of neighbouring countries, rabies will continue to encroach on rabies-free areas or areas where disease elimination is underway.The results of this study do not reflect negatively on the competency of the laboratory (CVL), but rather highlight the technical challenges faced in developing countries. These infrastructural challenges can affect the condition of samples during or after shipment and storage. Considering time and cost-effectiveness, accuracy and ease of use, the DRIT continues to demonstrate its potential value as a key player in the quest to improve rabies surveillance in the developing world.


The aim of the study was to describe the prevalence, incidence and persistence of depressive symptoms over a 36-month follow-up period among older people receiving in-home care, and to explore the association between cognitive function and the course of depressive symptoms. In all, 1001 older people (≥ 70 years) receiving in-home care were included in a longitudinal study over 36 months. Depressive symptoms, cognitive function, general medical health, activities of daily living, neuropsychiatric symptoms and use psychotropic drugs were assessed at three assessments. Dementia and mild cognitive impairment were diagnosed at all assessments. Baseline demographic characteristics and information on nursing home residency at follow-up were recorded. Linear mixed models were estimated. We found the prevalence and cumulative incidence of individual depressive symptoms to be higher in those with dementia at baseline than in those without. The persistence of depressive symptoms did not differ between those with or without dementia at baseline. The severity of cognitive decline and mean depressive symptom score assessed simultaneously were positively associated, but the strength of the association changed over time and was not significant at the last assessment. In conclusion: The differences in prevalence and cumulative incidence of depressive symptoms in those with and without dementia at baseline, and the association found between degree of cognitive decline and depressive symptoms over time shows that depression and dementia are interconnected. Nurses and clinicians should pay attention to cognitive status when observing or evaluating depression among older people receiving in-home care.


The current physical goods economy produces materials by extracting finite valuable resources without taking their end of the life and environmental impact into account. Mycelium-based materials offer an alternative fabrication paradigm, based on the growth of materials rather than on extraction. Agricultural residue fibres are inoculated with fungal mycelium, which form an interwoven three-dimensional filamentous network binding the feedstock into a lightweight material. The mycelium-based material is heat-killed after the growing process. In this paper, we investigate the production process, the mechanical, physical and chemical properties of mycelium-based composites made with different types of lignocellulosic reinforcement fibres combined with a white rot fungus, Trametes versicolor. This is the first study reporting the dry density, the Young’s modulus, the compressive stiffness, the stress-strain curves, the thermal conductivity, the water absorption rate and a FTIR analyse of mycelium-based composites by making use of a fully disclosed protocol with T. versicolor and five different type of fibres  and fibre processings . The thermal conductivity and water absorption coefficient of the mycelium composites with flax, hemp, and straw have an overall good insulation behaviour in all the aspects compared to conventional materials such as rock wool, glass wool and extruded polystyrene. The conducted tests reveal that the mechanical performance of the mycelium-based composites depends more on the fibre processing , and size than on the chemical composition of the fibres. These experimental results show that mycelium-composites can fulfil the requirements of thermal insulation and have the potential to replace fosile-based composites. The methology used to evaluate the suitability and selection of organic waste-streams proved to be effective for the mycelium-material manufacturing applications. The construction sector in Europe accounts for about half of all our extracted materials and energy consumption and for about a third of our water consumption and waste generation . InsteadThe development and implementation of this material in the field of architecture and construction has hardly been investigated and characterised so far . AdditioT. versicolor is known to be a non-selective white-rot, it removes lignin and structural carbohydrates  at a similar rate , wd = oven dry weight [g]  was used to measure the thermal conductivity according to ASTM D 5334–00 . The the1, 2… = initial and final temperature of the linear portion [°C], t1, 2… = initial and final time of the linear portion [s]Q = constant current [W/m], TThe method for Measurement of Rate of Absorption of Water by Hydraulic-Cement Concretes ASTM C 1585  with part = changed weight of the specimen [g], a = the exposed cross-section area [mm2], d = the density of the water [g/mm3].m = water absorption [mm], MFor the Fourier Transform Infrared Spectroscopy all IR spectra were acquired on a Nicolet 6700 FT-IR spectrometer from Thermo Fischer Scientific. The FTIR instrument was equipped with an IR source, DGTS KBr detector and KBr beamsplitters and windows. FTIR spectra were recorded in single bounce Attenuated Total Refractance (ATR) mode using the Smart iTR accessory, equipped with a diamond plate (42° angle of incidence). The spectra were recorded with automatic atmospheric correction for the background.-1 with 64 scans per sample. Four samples of each type were measured to ensure the reproducibility of obtained spectra . Compared to previous research, the water absorption is very low thanks to the two-phase fabrication method of the samples and the hydrophobic nature of mycelium. The study demonstrates that the manufacturing process impacts the desirable properties of the composite: the water absorption was much lower compared to previous studies thanks to the growth of a denser outer hydrophobic mycelial layer. However, other properties related to the thermal performances of insulating materials, such as fire resistance [Although the mechanical properties are not optimal yet, this research has shown that mycelium-composites can fulfil the requirements for thermal insulation foams and have the potential to replace fosile-based composites. The thermal conductivity and water absorption coefficient of the mycelium composites with flax, hemp, and straw have shown an overall good insulation behaviour in all the aspects compared to conventional insulation materials such as rock wool and glass wool . The hemsistance , aging, T. versicolor has a small preference for hemicellulose over cellulose. The relative presence of chitin was not influenced by the type of substrate. The increase in ratio of peak intensities between decayed and undecayed flax fibres reveals a higher intensity of lignin band of decayed flax than undecayed flax. The depolymerisation of lignin by T. versicolor happen to a greater extend in flax than in hemp.The ratios of peak intensities of the FTIR spectra  revealedGenerally, the conducted tests reveal that the mechanical performances of the mycelium-based composites depend more on the fibre condition, size, processing, than on the chemical composition. The methology used to evaluate the suitability and selection of organic waste-streams proved to be effective for the mycelium-material manufacturing applications. The wide spectrum of options to compose and grow mycelium-composites makes it complex to compare the results with existing literature since every change of variable affects the growth and mechanical behaviour of the composite. Further work is required to improve the growth conditions, to optimize the mechanical properties and to establish a standard fabrication protocol.S1 Fig(a) mycelium chitinous layer, (b) air-void, (c) limited decayed fibre by mycelium.(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 FigThe mycelium composites had a lower thermal conductivity than water, therefore lower electrical current was used to mark a clear increase of temperature during the test. The temperature changes depending on the applied current during the test. The response of the probe’s temperature is monitored in function of the time. Nonetheless, as expected, the overall results of the calculations did not present large differences. The higher the applied current, the faster the temperature increased, and thus the lower the thermal conductivity. Yet, this fast rise in temperature is not recommended by the standard due to possible errors while readings. Therefore it was more reliable to take the average of the values for thermal conductivity corresponding to a lower applied current.(TIF)Click here for additional data file.S4 Fig(TIF)Click here for additional data file.S1 TableA different electrical current in the circuit was applied for every sample.(DOCX)Click here for additional data file.


Many of the industrial processes, including material removal operation for shape generation on the surface of material, exploit the assistance of ultrasonic vibrations. This trend of using ultrasonic vibration in order to improve the process performance is becoming more and more prominent recently. One of the significant applications of this ultrasonic vibration is in the industrial processes such as Electro-discharge machining (EDM), where ultrasonic vibration (UV) is inserted as a medium for enhancing the process performance. Mostly ultrasonic vibration is applied along with the EDM process to increase the efficiency of the process through debris cleansing from the sparking gap. There have been significant changes in ultrasonic assisted technology during the past years. Due to its inherent advantages, ultrasonic assistance infiltrated in different areas of EDM, such as wire cut EDM, micro EDM and die sinking EDM. This article presents an overview of ultrasonic vibration applications in electric discharge machining. This review provides information about modes of UV application, impacts on parameters of performance, optimization and process designing on difficult-to-cut materials. On the bases of available research works on ultrasonic vibration assisted EDM, current challenges and future research direction to improve the process capabilities are identified. Literature suggested improved material removal rate (MRR), increased surface roughness (SR) and tool wear ratio (TWR) due to the application of ultrasonic vibration assisted EDM. However, tool wear and surface roughness can be lessened with the addition of carbon nanofiber along with ultrasonic vibration. Moreover, the application of ultrasonic vibration to both tool and workpiece results in higher MRR compared to its application to single electrode. Electric discharge machining (EDM) is a non-conventional process based on electro-thermal energy and is used for machining of diverse materials with complex geometrical shapes as well as precise dimensional cuttings. EDM process is especially well recognized for manufacturing of hard-to-machine materials. By overcoming technical difficulties associated with the conventional machining, EDM technology finds its appropriate applications not only in the aerospace & automotive industries but also in the surgical and medical implant creation ,2. In adDuring the EDM process, when voltage between two electrodes is sufficiently high enough to break the dielectric strength, pulse discharge current generated between tool and workpiece aids in materials removal. Melting and vaporization of specimen material are caused by the dielectric liquid breakdown. The removed debris materials gather in the machining gap which eventually decreases the resistance in this gap . ConsequThe general purpose of the article is to unfold the underpinning knowledge of Ultrasonic vibration assisted EDM process, its performance parameters, modes of applications, process modelling, metallurgical characteristics on the surface and optimization of the process.Electrical discharge machining (EDM) is the method of removing electrically conductive materials in the form of small craters ranging from several to tens of microns using strictly coordinated sparks occurring between a tool electrode and a workpiece in the presence of a dielectric fluid . The mecThe sparking and gap phenomena in EDM and micro-EDM are comprised of three main phases, preparation for the sparking to occur, sparking phase itself and interval phase between two consecutive sparking phases . At the One of the major challenges of EDM and micro-EDM is the low throughput/MRR. This challenge becomes even more evitable during the micro scale machining of blind features. As the UV assisted EDM combines the effect of ultrasonic cleaning at the narrow spark gap of few microns with the traditional discharging induced material removal, the overall MRR increases. This is mainly due to two reasons. The ultrasonic vibration induces cavitation bubbles into the dielectric liquid at the spark gap, resulting in micro jets, which aids in removal of debris from the machined zone effectively. In addition, due to the instantaneous and continuous removal of debris and circulation of fresh dielectric into the spark gap, the discharge frequency increases, which further increases the MRR.Another challenge in EDM and micro-EDM is the machining instability due to very small spark gap (in the range of microns), resulting in a long machining time, high electrode wear and surface defects. During machining of deep holes or complex 3D microstructures using die-sinking micro-EDM, removal of debris from the small spark gap becomes challenging. The debris, if not removed instantly and efficiently, creates a bridge of debris particles resulting in a short circuiting between the workpiece and the electrode, thus ceasing the machining operations. In addition, due to the higher concentration of debris particles in the small spark gap, the arcing (continuous sparking) occurs on the debris particles causing the adhesion of debris particles on the machined surface and resulting in surface and sub-surface defects. Ultrasonic vibration assisted EDM has the capability of solving those two major challenges in EDM and micro-EDM, thus enhancing the capability of EDM in deep hole micromachining and complex 3D microstructures. The UV assisted EDM can also be useful in machining of large 3D structures using conventional die-sinking EDM and high aspect ratio and thin slot cutting using wire EDM.Ultrasonic vibration (UV) assisted EDM/micro-EDM is a hybrid process that combines ultrasonic vibration to the workpiece, electrode or dielectric during the conventional EDM or micro-EDM process. In this process, the ultrasonic vibration (frequency of 20 kHz or above) is introduced during the EDM process to improve the flushing efficiency of the process. Based on the applications and the challenges faced during the EDM/micro-EDM process, the ultrasonic vibration can be applied to the tool electrode , workpieIn the tool vibration assisted EDM, the ultrasonic vibration is applied to the tool electrode through a high frequency piezoelectric transducer that is connected to the tool electrode through a holder See a. The ulIn this system, the ultrasonic vibration in the form of sinusoidal wave with desired frequency are generated by the pulse generator and transmitted to the piezoelectric transducer (PZT). The PZT actuator is connected to the workpiece through a joint plate, which transfers the ultrasonic vibration to the workpiece, as shown in The ultrasonic vibration can also be applied to the dielectric fluid to create the turbulence into the small spark gap between the electrode and the workpiece, and effectively flush away the debris from the machined zone c. SeveraThe ultrasonic vibration assisted EDM in gas medium is basically a hybrid process that combines the ultrasonic vibration to the workpiece during dry EDM (or EDM with gas as a dielectric). In dry EDM, high pressured gas is used as a dielectric fluid that is supplied mainly through the centre of the tool electrode during the EDM process. In dry EDM, short circuiting happens frequently due to the narrow spark gap of few microns and comparatively higher applied voltage. As a result, in order to enhance the machining stability and MRR, the ultrasonic vibration is applied to the workpiece in addition to tool rotation. Both the tool rotation and ultrasonic vibration helps in reducing the short-circuiting and thus enhancing the machining stability. The ultrasonic vibration further facilitates the removal of molten material and resolidified debris from the workpiece and the narrow spark gap, thus generating the sparking on the fresh surface of the workpiece and improving the MRR. There have been several studies attempting to model ultrasonic vibration assisted EDM, both in liquid and gas dielectric mediums. One of the early studies on the UV assisted EDM in liquid dielectric by Zhang et al. (2006)  developeM = MRR, totV = summation of the volume removed by all single sparks, totT = total time duration, η1 = proportional constant, eI = discharge current, eU = discharge voltage, it is the idling time, st is the pulse duration.The analytical equation derived for calculating the MRR is as follows :(1)M=VtoThe equation can be further simplified for the assumption that all the discharge pulses are square, as shown in totV, they were able to calculate/estimate the values of average surface roughness aR.  assumed that the crater formed by a single spark is of spherical crest size with radius R and crater depth H, as shown in ness Ra. b The ana follows :(3)Ra=ξ1MRR = material removal rate, V = summation of the volume of material removed by all single sparks, to is discharge pulse-on time and dt is pulse-off time, η is a coefficient. ρ is Density, f is the frequency and a is the amplitude of ultrasonic, T = total time duration, η1 = proportional constant, eI = current, eU = discharge voltage, andThe same research group, in a later study led by Xu et al. (2010) further modified the equation of MRR for the UV assisted EDM in gas. Considering the same geometry of the crater, spherical crest, as shown in D and crater height H, the following constant was simplified in the model.And for crater diameter Shabgard et al. (2017)  carried NumRLT) has been calculated as follows:cD = Depth of crater and %PFE = plasma flushing efficiencyThe numerical value of recast layer thickness (CV(FEM) is the theoretical volume of melted material per pulse and CV(EXP) is the experimental volume of removed material per pulse.The plasma flushing efficiency can be obtained as:CV(EXP)) is obtained by:M1 and M2 are the weight of workpiece before and after machining, respectively, NN is the number of normal pulses at the end of machining period and ρ is the workpiece material density.The experimental volume of removed material per pulse   establism = mass of the molten metal droplet, maxa = y″max = maximum acceleration of the molten drop, where f is the frequency of ultrasonic vibration, A is the amplitude of ultrasonic vibration, t = time.The developed equation for the maximum inertia force of the molten metal drop is as follows:f is the frequency of ultrasonic vibration, A is the amplitude of ultrasonic vibration, D is the diameter of the discharge crater and H is the depth of the discharge crater.The maximum inertia force for the crater can be calculated as:M is a coefficient, ρ is the density of the metal, mT is the melting point of the metal, C1 is adjusting coefficient of density and C2 is adjustment coefficient of melting point.And the surface tension of the liquid drops can be obtained by the following formula :(14)σa=1From the above three equations, it was easily determined that melting liquid drops could be easily shot off by the effect of inertia force caused by ultrasonic vibration, thus improving the effectiveness of the ultrasonic vibration assisted EDM.Various types of UV assisted EDM have been reported in the literature. These studies can be classified into three categories, such as, vibration to tool/wire, vibration to workpiece and vibration to dielectric. This section presents the summary of research studies carried out on these three kinds of UV assisted EDM.For achieving better performance, tool horn should match the resonant frequency of the transducer a. Nanu eIn the research of Hirao et al. , the appShervani-Tabar et al.  investigOn another study, Shervani-Tabar et al.  also invZhang et al.  also rep3N4, it is important that the stray conductive layer remains on the ceramics surface to continue the EDM process, however Mohri et al. reported on the disturbance in the generation of this conductive layer due to the tool vibration [In order to improve the MRR of insulating ceramics associated with EDM process, Praneetpongrung et al.  investigibration . Therefoibration  suggesteibration  have invGhoreishi et al.  studied 2O3 was machined using UVA-EDM and their results suggested increased MRR when compared with the summation of MRR by UVA, EDM, however, increased MRR also enhances generated surface roughness. Increase of the discharge power or voltage, vibration amplitude and frequency aid in MRR due to the efficient discharge distribution and easier debris removal. Uhlmann et al. [In another study done by Lee et al.  insulatin et al.  studied n et al. .Lin et al.  studied Zhixin et al.  proposedSrivastava et al. ,57 compaIn order to avoid the environmental hazard of dielectric fluid, researchers proposed dry EDM mechanism, however, this process has limitations in terms of poor surface quality due to the inefficient flushing. Xu et al. ,59 invesDeep hole fabrication also comes with the taperness effect. Kim et al.  used resHuang et al. ,67 demonMastud et al.  investigOther than, EDM, micro-EDM; as well as wire-EDM plays a significant role for hard to cut material fabrication. Several studies reported on the improvement of process by applying vibration assisted WEDM. Guo et al.  demonstrHoang et al.  investigMohammadi et al. ,78 conduWen-Jeng Hsue1  investigUnne et al.  studied Prihandana et al.  also conJiang et al.  developeTeimuri et al.  studied Fabrication of non-circular micro-structures always has difficulties in terms of debris evacuation due to narrow discharge gap, therefore, machining efficiency can be significantly low. Tong et al.  applied 3 s−1. The machining depth increases with the increase of amplitude and frequency, where frequency is the most dominant parameter. In addition, slower scanning speed also enhances the dwelling time in the favourable discharge range, therefore, aids in improving the discharge ratio as well as stable machining. Fu et al. [Hao et al.  proposedu et al.  developeu et al.  reportedJahan et al.  derived Garn et al.  studied Gao et al.  reportedPrihandana et al.  also inv2 powder, showed a considerable increase in material removal rate , the surface roughness (SR) and tool wear rate (TWR).MRR associated with ultrasonic vibration assisted EDM is always higher than that with pure EDM. As can be seen from UV assisted EDM process not only increases the MRR due to the application of UVA but also increases the roughness of the generated surface a. For thNevertheless, Guo et al. reported on reduced surface roughness for UVA-WED b. The reThe electro-discharge machining of ceramics became popular among the metal cutting industries because of its features and competitive cost. Lee et al.  studied Titanium alloy Ti–6Al–4V) is one of the hardest materials to cut using conventional machining processes. Ultrasonic vibration assisted EDM has been studied for machining this alloy . MateriaAl–4V is 3N4 was investigated using EDM combined with ultrasonic vibrations [Effect on machining properties of Sibrations . It is rbrations . When shbrations  investigbrations  investigThere has been significant amount of works done on the feasibility studies of the UV assisted EDM using different dielectric mediums and applying vibration onto the workpiece, tool and dielectric. Many of those studies focused on investigating and comparing the performance of UV assisted EDM/micro-EDM with the traditional EDM/Micro-EDM. In addition, a significant amount of works has been carried out on studying the effect of both electrical and vibration parameters on the machining performance parameters such as MRR, tool wear and surface finish. However, a number of research questions remain unanswered, that should be considered by the future researchers as scopes for future research. Following are some suggestions for the future research on this area of EDM.One of the issues in UV assisted EDM or micro-EDM, is the accuracy of the parts or structures produced. This issue is more important to be considered in tool vibration assisted EDM, however, it was also found to cause inaccuracies in workpiece vibration assisted EDM. Due to the ultrasonic vibration, the spark gap changes continuously, which results in changes of overcut by the process. As a result, an irregularity in the machined hole diameters or difficulty in achieving sharp corners in the microstructures were observed. Future research should try to resolve those issues and investigate the ways of improving the part accuracy, while enhancing the MRR.Although there have been studies on investigating the surface topography and roughness generated in UV assisted EDM, in depth studies investigating the sub-surface microstructure and mechanical properties changes of the part machined by the process is still insufficient. In addition, for most of the cases UV assisted EDM generated higher surface roughness compared to traditional EDM process. Therefore, future research should focus on investigating the sub-surface microstructural changes after conventional EDM and UV assisted EDM to see if the application of ultrasonic vibration helped in minimizing the heat affected zone at the sub-surface and improve the microstructure at the sub-surface level. In addition, various mechanical properties including residual stress, tensile and fatigue properties of the UV assisted EDM process machined parts can be compared with the traditional EDM process machined parts.The current research on UV assisted EDM lacks from the sensing, monitoring and control of the process, which could possibly resolve the inaccuracy issue discussed in the previous point. Very few research studies have focused on this important issue. The in-situ monitoring of the sparking conditions at the spark and/or the width of the plasma channel and feedback to the control system to control either the electrode feed or vibration frequency/amplitude to maintain the stable plasma column width, will be able to improve the dimensional accuracy in UV assisted EDM/micro-EDM.The tool wear compensation during the UV assisted EDM can also improve the dimensional accuracy of the parts machined. There have been very few research studies on monitoring the tool wear and developing algorithms for tool wear compensation in UV assisted EDM, although there were many studies on the same for conventional EDM and/or micro-EDM. The authors believe that it is more important to monitor and compensate tool wear in UV assisted EDM to maintain the accuracy of the parts and structures.There have been very few studies on modelling the UV assisted EDM process in both liquid and dielectric mediums. Several studies have developed the models of MRR and SR based on the geometry of the crater sizes and Gaussian heat source without considering the ultrasonic vibration parameters, such as frequency, amplitude and vibration induced cavitation. It is important to note that unlike conventional EDM, the cavitation is a major physical phenomenon that should be considered during the modelling of the UV assisted EDM or micro-EDM. Therefore, future research should focus on more physics-based modelling considering the cavitation as secondary material removal phenomenon in addition to sparking induced craters. Moreover, the effect of vibration frequency and amplitude should be taken into consideration when modelling the UV assisted EDM or micro-EDM.In recent years, there have been a few studies that focused on integrating assistance of other physical phenomena with the ultrasonic vibration assisted EDM, such as UV assisted EDM with powder mixed dielectric, where more than one physical assistance was used. Future research studies should explore more on this area of hybrid machining by including the assistance of other physical principles with UV assisted EDM to further improve the machining performance of the conventional EDM process. Some examples can be combined magnetic and UV assisted EDM, UV assisted electro-chemical discharge machining (UV assisted ECDM), UV assisted EDM-ECM combined process, combined UV and laser assisted EDM and so on. A lot of research opportunities will open up with the development of new hybrid machining processes as suggested above, including initial feasibility study, performance characterization and optimization, modelling of the process and applications of the new process, to name a few.Finally, the application of UV assisted EDM and micro-EDM process could be broadened to semiconductor and micro and nanoelectromechanical systems (MEMS/NEMS) by investigating the machinability of new and innovative materials using the UV assisted EDM process. One of the major challenges in UV assisted EDM of semiconductor materials like silicon or germanium wafer is the recast layer formation and heat affected zone beneath the machined surface, that modify the electrical and thermal properties of the semiconductor materials. As UV assisted EDM/micro-EDM is found to minimize the recast layer thickness underneath the machined surface, UV assistance can be used with the wire EDM or micro-EDM to cut semiconductor wafers or machine 3D microstructures with semiconducting materials without making changes in important electro-thermal properties. Extensive research on this issue is needed before UV assisted EDM/micro-EDM be an acceptable machining process in the semiconductor industries.Ultrasonic assisted EDM is a hybrid process that combines the physical phenomena of ultrasonic vibration with the EDM and/or micro-EDM process to improve the process stability and performance. The ultrasonic vibration was found to be applied effectively in both workpiece and tool electrode based on the applications and structures to be machined. This review paper provides an overview of the principle of EDM and ultrasonic vibration assisted EDM, as well as includes a comprehensive review of various aspects of ultrasonic vibration assisted EDM including major challenges and guidelines to resolve the challenges in the future research. It can be concluded from the literature that ultrasonic vibration assistance increases the MRR, surface roughness and tool wear ratio due to the reduced arcing, inactive pulses, cavitation effect and stable discharge. The MRR can be enhanced by increasing voltage, current and vibration amplitude. Increase of discharge voltage, current, pulse duration and ultrasonic vibration amplitude causes increased surface roughness, whereas higher frequency vibration results in a lower surface roughness due to the generation of multiple channel of discharge. On the other hand, TWR can be reduced by increased vibration amplitude, tool rotation and planetary affect. The ultrasonic vibration assisted EDM was found to be especially effective, where the flushing of debris becomes challenging, such as in deep micro-holes. The aspect ratio and inside surface quality of the micro-holes could be significantly improved by applying ultrasonic vibration in micro-EDM drilling of deep micro-holes. Ultrasonic vibration assisted EDM was also found to be effective during the machining of complex blind structures for multi-scale die and mould making. The ultrasonic vibration assisted EDM was also found to improve the surface integrity by reduction of the ineffective pulses, that is, arching and short-circuiting, on the surface, thus reducing the thickness of recast layer and residual stress. Finally, it can be concluded that ultrasonic vibration assisted EDM is an effective process to machine difficult-to-cut materials and complex multi-scale structures on a range of materials including metals and ceramics, thus, improving the process capability of the traditional EDM and micro-EDM process.


These inhibitors can provide novel tools for basic research and can serve as starting-points to develop new chemotherapeutics.The formation of new ribosomes is a fundamental cellular process for each living cell and is tightly interwoven with cell cycle control and proliferation. Minimal disturbances of this pathway can result in ribosomopathies including an increased risk for certain cancer types. Thus, targeting ribosome biogenesis is an emerging strategy in cancer therapy. However, due to its complex nature, we are only at the beginning to understand the dynamics of the ribosome biogenesis pathway. One arising approach that will help us to embrace the tight timely cascade of events that is needed to form a new ribosome is the use of targeted chemical inhibition. However, only very few specific chemical inhibitors of the ribosome biogenesis pathway have been identified so far. Here we review our recently published screen to identify novel inhibitors of the ribosome biogenesis pathway in yeast (Awad  Ribosomes are ribonucleoprotein complexes in the MDa range and translate the genetic code into proteins. Hence, the efficient production of new ribosomes is essential for every growing and dividing cell. In eukaryotic cells, ribosome biogenesis requires around 250 non-ribosomal assembly factors and ranks among the most energy and resource demanding processes. Ribosome formation is linked with other fundamental circuits controlling cell cycle and proliferation through a tight regulatory network. Therefore, dysregulation of ribosome biogenesis in any direction (up- or downregulation) affects cell proliferation rate and can favor the unleashed growth of tumors or result in developmental disorders, anemia or bone marrow failure . Fast proliferating cells, including cancer cells, strongly depend on high levels of ribosome biogenesis, whereas quiescent cells can presumably survive a temporary shutdown of the pathway with their available depot of mature ribosomes. Accordingly, the strong dependency on high levels of ribosome biogenesis represents an Achilles heel of cancer cells and its inhibition represents a promising strategy for the treatment of tumors or infections.Eukaryotic ribosomes are composed of ~80 ribosomal proteins and four rRNAs that have to be thoroughly assembled in a sophisticated cellular manufacturing line. Nascent ribosomes start their maturation in the nucleolus, where the rRNAs are transcribed and rapidly loaded with the first assembly factors. The mature rRNAs are then carved out of the raw RNA precursor transcript in numerous processing steps. Cleavage at a specific site of the primary transcript gives birth to the precursors of the two ribosomal subunits, the pre-40S and the pre-60S particles, which are further assembled in two independent pathways. While the pre-40S precursor is rapidly transported into the cytoplasm, where the final maturation takes place, the pre-60S particle undergoes numerous rearrangements and rRNA processing steps until it is exported into the cytoplasm for finalization.Despite the tremendous progress in our understanding of ribosome biogenesis in recent years, the full complexity and especially the dynamic nature of the maturation cascade still remain enigmatic and crave for novel approaches. Therefore, it is of great importance to understand the cascade of events during ribosome biogenesis and its regulatory network in detail. In the past, common genetic and biochemical studies, mostly done in yeast, gathered basic knowledge about ribosome formation including groundbreaking structural insights. However, the available tools provide rather static snapshots and hence are not suitable to unravel the dynamics of this rapid and interwoven pathway. One promising strategy to circumvent these limitations is the use of small molecular weight inhibitors that allow experimenters the rapid blockage of ribosome formation at a distinct step and to monitor the effects within minutes.However, despite the large repertoire of potential targets provided by the ~250 maturation factors alone, only very few chemical inhibitors of ribosome biogenesis are known so far. With the help of diazaborine, the first inhibitor of eukaryotic ribosome biogenesis, our lab previously demonstrated the tremendous potential to investigate the dynamics of ribosome biogenesis by targeted chemical inhibition.By inhibiting the AAA-ATPase Drg1 in the cytoplasm, diazaborine specifically blocks release and recycling of shuttling pre-60S maturation factors. Consequently, these shuttling factors are trapped on cytoplasmic particles, which leads to their depletion in the nucleus. The resulting shortage for the next round of ribosome biogenesis in the nucleus and can lead to secondary blocks, eventually causing entrapment of even earlier acting maturation factors. This rebound effect is therefore transmitted back step by step to the earliest stages of pre-ribosome formation. Since there is hardly any free pool of maturation factors, these secondary and tertiary blockages manifest within minutes. Unraveling this cascade of events was a crucial step to understand the coordination of nucleolar and cytoplasmic maturation events and demonstrated that the full dynamics of ribosome biogenesis can only be disentangled with the fast onset of chemical inhibition.in vivo screen . Our screening approach is based on the fact that perturbance of ribosome biogenesis often causes an entrapment of pre-ribosomal particles in the nucleoplasm or the nucleolus. Thus, to score for ribosomal particle maturation defects, we separately GFP-tagged selected ribosomal reporter proteins for the small and the large subunit. Since these ribosomal reporter proteins are components of mature ribosomes, the GFP-signal in untreated cells is located in the cytoplasm. Accordingly, a shift of the fluorescence signal to the nucleolus and/or nucleoplasm upon inhibitor treatment indicates a block in ribosome biogenesis. This simple basic setup allowed us to rapidly screen ~1000 compounds, an approach, which could be extended to larger compound collections.In order to find new tools for targeted chemical inhibition, we systematically screened collections of naturally occurring and/or clinically used substances for inhibitory effects on ribosome biogenesis in yeast using a microscopy-based In the frame of this study, we identified 128 compounds inhibiting maturation of either the small or the large ribosomal subunit or both (indicative of a very early block in the pathway). Northern blot analysis demonstrated a broad spectrum of different rRNA processing defects caused by these inhibitors, indicating that the compounds affect a wide range of different maturation steps within the pathway. While some of the identified substances were already known to interfere with the RNA metabolism (e.g. Acivicin and Mycophenolic acid) or had been linked to ribosome biogenesis before (e.g. Vulpinic acid), the vast majority of compounds were for the first time recognized as inhibitors of the ribosome biogenesis pathway. Strikingly, many of the inhibitors identified in the screen were shown previously to affect tumor cell growth or had already been in clinical use for cancer treatment (e.g. Carmofur or Rubicin derivatives). This result suggests that ribosome biogenesis is an extremely promising pathway for antitumor chemotherapy and provides a rich source of novel inhibitor targets to be explored in future studies.In sum, the work from Awad et al., 2019 delivers a first comprehensive set of ribosome biogenesis inhibitors to dissect the dynamic character of this pathway in basic research, but also establishes ribosome biogenesis as promising target for drug design.


Human-induced pluripotent stem cells (hiPSCs) provide a personalized approach to study conditions and diseases including those of the eye that lack appropriate animal models to facilitate the development of novel therapeutics. Corneal disease is one of the most common causes of blindness. Hence, significant efforts are made to develop novel therapeutic approaches including stem cell-derived strategies to replace the diseased or damaged corneal tissues, thus restoring the vision. The use of adult limbal stem cells in the management of corneal conditions has been clinically successful. However, its limited availability and phenotypic plasticity necessitate the need for alternative stem cell sources to manage corneal conditions. Mesenchymal and embryonic stem cell-based approaches are being explored; nevertheless, their limited differentiation potential and ethical concerns have posed a significant hurdle in its clinical use. hiPSCs have emerged to fill these technical and ethical gaps to render clinical utility. In this review, we discuss and summarize protocols that have been devised so far to direct differentiation of human pluripotent stem cells (hPSCs) to different corneal cell phenotypes. With the summarization, our review intends to facilitate an understanding which would allow developing efficient and robust protocols to obtain specific corneal cell phenotype from hPSCs for corneal disease modeling and for the clinics to treat corneal diseases and injury. Isolation of human embryonic stem cells (hESCs) from the inner cell mass of a human embryo  initiateCorneal diseases are the most common debilitating source of visual loss that may lead to permanent blindness . AlthougThe cornea provides two thirds of the refractive power of the eye and is composed of five well-defined layers Fig. , includiThe protocols devised for differentiating pluripotent cells to a particular cell fate has mostly relied on the developmental studies of the particular cell or organ in question. In case of the eye, our knowledge is mostly derived from the developing mouse  and chicThe pathologies of most of the corneal diseases including the dystrophies are usually limited to a specific layer of the cornea . Replace2 was conductive to the differentiation of CE progenitors from hESCs [The integrity and homeostatic function of the corneal epithelium are crucial for maintaining the transparency and visual function of the cornea. Under homeostasis conditions, the corneal epithelium (CE) is renewed and maintained by its progenitor cells in the limbus. An injury or disease causing the loss of CE affecting corneal health and its function has therefore been a matter of interest in the ocular field. To treat the loss of CE, current therapies involve direct implantation of the limbal tissue containing LESC population from the unaffected eye when the complication is unilateral. However, limitations to such transplantation therapies arise from the risk of damaging the donor healthy eye from which the LESCs are obtained in the case of unilateral transplantations . While iom hESCs . Howeverom hESCs . Directeom hESCs  while CKom hESCs . In ordeom hESCs , with doom hESCs . TherefoKeratoplasty is a primary treatment option to treat many of the corneal conditions including corneal injury, corneal dystrophy, keratoconus (KC), and corneal infractions . The eveThese studies highlight the necessity for stepwise paradigms, where the iPSCs are first driven to the intermediate neural crest (NC) stage followed by a robust directed differentiation to CSKs. An interesting attribute of culturing CSKs which has been applied in devising iPSC-based protocols to derive CSK is their ability to form aggregates and maintain their phenotype when deprived of substratum attachment . Funderb2 post-natal to 2000/mm2 in older adults [Human corneal endothelium (hCEn) which originates from cranial NC cells is approximately 4 μM in thickness. This monolayer of hexagonal hCEn cells lining the Descemet’s membrane of the posterior cornea maintains the dynamic fluid and nutrient balance across the stroma . Being hr adults , 79. As r adults . Additior adults . The hPSr adults  introducr adults  reportedr adults  demonstrr adults  reportedr adults . McCabe r adults  followedr adults  have delr adults , 89. Sucr adults , 91. iPSr adults ). The bir adults  and expar adults , 94  canThough iPSC technology has huge potential for regenerative medicine and disease modeling, it faces many challenges and limitations which require further in-depth understanding of the cellular reprogramming differentiation processes Table . One of The iPSCs and corneal cells differentiated from them have a significant risk of genomic instability due to the extended in vitro culture periods required , 113. GeUse of iPSC-derived corneal cells in the clinical application has multiple challenges, which includes derivation of clinical grade cells, potential tumorigenicity of transplanted cells, and immune-acceptance of transplanted cells. However, there are inherent advantages and disadvantages considering autologous or allogenic iPSC-derived corneal cells for cell replacement therapy. By passing the issue of immune rejection is the primary advantage of the autologous iPSC-derived corneal cells. However, the crucial challenge that would need to be addressed for the utility of such truly autologous iPSC-derived corneal cells is the time required to generate such individual iPSC line. The iPSC generation time which ranges in terms of weeks to months depends on the multitude of factors such as the age of the donor, phenotype of the somatic cell to be re-programmed, reprogramming method, and culture conditions. Additional expenditure of time and cost will be needed for the selection and characterization of the individual iPSC clones, and their derivatives significantly increasing the cost of therapy. A special advantage with most of the corneal diseases being chronic in nature is the availability of sufficient time to strategize and perform the necessary processes for generating corneal cells from autologous iPSCs. However, it is important to note that autologous cells carrying gene defects will need to be corrected necessitating more time and cost for the process and characterization of the derived cells and to be taken into consideration while devising strategies for the generation of corneal cell phenotypes from iPSCs. For accessibility and application of iPSC-based cell therapy, it is important to address the challenge for keeping the costs affordable yet have a robust derivation process of corneal cells within an acceptable time line. The Japanese study  using auAnother aspect which favors the proponents of allogenic PSC source for generating corneal cells is the eye being considered an immune-privileged site due to its relative self-containment due to the barriers that keep cells from migrating both from inside or outside to other parts of the body. With progress in the field providing us with deeper insights into the mechanisms of cellular reprogramming and their induction to specific corneal cell lineages and the stability of the phenotypes will allow surmounting the concerns and paving possibilities for their utilization in the clinics.Though iPSC technology has huge potential for disease modeling, it faces many challenges  which maHere, it is important to note that the epigenetic changes between the somatic cells and the derived iPSCs may obscure the retention of the disease phenotype for disorders that involves epigenetic modification such as imprinting disorders or sex-linked disorders or for diseases with mixed etiologies. One potential limitation is the genetic variability between different patients or clones derived from the same patient  which caDerivation of iPSCs and differentiation to corneal cell types in a personalized manner would be an asset to venture upon in order to achieve customization of patient-specific therapy. Though the cost of multiple quality control parameters at key points of the iPSC derivation, clone characterization, and differentiation process remain expensive, the establishment of a robust technique along with the development of commercialization of “kit”-based tools can make the process affordable. Personalized iPSC-derived therapy for corneal tissue replacement is underscored by the presence of pathological genetic mutations that can be addressed by constructing population-based mutation databases with strong clinical phenotype correlation. This would eliminate the mutation screening step to some extent thereby moving forward with gene editing step. The limitation of personalized iPSC-based cell therapy for corneal diseases can be circumvented by a slightly different and perhaps logistically and financially less burdensome strategy of developing universal donor iPSC lines which can be immune-match up for a higher percentage of the population . In presTo conclude, despite these promising results, more research is needed for understanding and addressing the risks involved in using cells de-differentiated from iPSCs which include right from the process of iPSC generation to its differentiation and its later utilization. A tri-party amalgamation involving the researcher, clinician, and an industry partner would achieve for providing affordable and reproducible results in patients with corneal diseases. Here, we provide a review of the application of the iPSC technology to generate corneal cell phenotypes for modeling corneal diseases and allow interrogating the genotype-phenotype relationship in a tissue-context manner. These insights would lead to the identification of possible newer molecular targets in the disease-causing pathway which can be modulated for therapy. Furthermore, in the near future, in vivo-corrected corneal cells from patient-derived iPSCs can find applications for cellular transplantation to address corneal diseases.Chambers et al.  and Lee


Rupture of the patellar tendon is relatively rare. We report a case of patellar tendon avulsion with a tibial tuberosity sleeve fragment in pediatric patient. In pediatric patient, diagnosis is sometimes difficult due to uncompleted ossification. In the present case, which involved the presence of a small fleck of bone from tibia, we were able to attain a diagnosis using the Koshino-Sugimoto index and MRI and easily determine the optimal treatment with the use of the suture anchor and tension band wiring method. Rupture of the patellar tendon is relatively rare. The rates of patellar tendon injury is 3% of all knee extension system injuries . In mostA 12-year-old boy was referred to our hospital with an acute injury of the left knee that had occurred while playing soccer. Slight swelling and pain at the knee were observed. Assessment of active range of motion was impossible due to pain. The patient had no history of chronic medication or Osgood-Schlatter disease. Radiographs and computed tomography (CT) showed small flecks of bone at the proximal tibial tuberosity . MagnetiThe main cause of extensor mechanism injuries of the knee is forceful contraction of the quadriceps against a partially flexed knee or firm resistance. The quadriceps undergoes an eccentric load avulsing the patellar tendon complex. Failure of the patellar tendon is relatively uncommon among extensor mechanism injuries of the knee , 5. ZernAs a disorder or injury that we should distinguish from our case, Osgood-Schlatter syndrome (OSS) and avulsion fracture of tibial tuberosity are common. OSS suffer the tibial tuberosity in growing children and presents with local pain, swelling, and tenderness of the tuberosity. X-ray shows fleck of bone at tibial tuberosity. However, it is not a traumatic disease but disorder like enthesopathy. Our case existed the history of trauma and no pain before injury. Moreover, while OSS does not show the obvious patella alta on X-ray, our case shows the obvious patella alta on X-ray. Of course, it is unclear whether OSS is one of the risk factor for our case. Our case may have the possibility of vulnerability cause of immature bone or asymptomatic OSS.Whereas the distinguishing between patella tendon rupture and avulsion fracture was difficult in the present case because the fleck of bone was tiny and the displacement was minimal. Desai and Parikh  reportedIn conclusion, when a pediatric patient sustains rupture of the patellar tendon from the tibial attachment, diagnosis is sometimes difficult due to uncompleted ossification. In the present case, which involved the presence of a small fleck of bone from tibia, we were able to attain a diagnosis using the Koshino-Sugimoto index and MRI and easily determine the optimal treatment.


In this research work we have examined the flow of Williamson liquid film fluid with heat transmission and having the impact of thermal radiation embedded in a permeable medium over a time dependent stretching surface. The fluid flow of liquid films is assumed in two dimensions. By using suitable similarity transformation the governing non-linear partial differential equations have been transformed into non-linear differential equations. An optimal approach has been used to acquire the solution of the modelled problem. The convergence of the technique has been shown numerically. The impact of the Skin friction and Nusslet number and their influence on thin film flow are shown numerically. Thermal radiation, unsteadiness effect and porosity have mainly focused in this paper. Furthermore, for conception and physical demonstration the entrenched parameters, like porosity parameter  The field of thin film flow problems is vast and is realized in many fields, starting from the particular situation of the flow in human lungs to lubricant problems in industry. Investigating the uses of thin liquid film flow is an interesting interaction between structural mechanics, fluid mechanics, and theology. Extrusion of polymer and metal, striating of foodstuff, constant forming, elastic sheets drawing, and fluidization of the devices, exchanges, and chemical treating apparatus are several well-known uses of liquid films. In observations of these uses and applications, the study of liquid film becomes necessary for researchers to further investigate and make further development in it. Different approaches with modified geometries have been adopted by many researchers from time to time. In view of the industrial applications of thin film flow, stretching surface has become an important topic for researchers. In early days, the study of liquid film flow was limited to viscous fluids. Crane Non-Newtonian fluids have so many types in nature as well as in artificial. Williamson fluid is one of significant subtypes between them. A number of researchers investigated Williamson fluid with different effects. Practical application has produced interest in searching the solvability of differential equation governing in flow of Non-Newtonian liquids, which have numerous uses in engineering field, applied mathematics and computer science. Many environmental and industrial systems like system of geothermal energy and system of heat exchanger design include the convection flow subject to permeable medium. The adapted form of classical Darcian model is the non-Darcian porous medium, which contains the inertia and boundary topographies. The standard Darcy's law is effective under constrained range of small permeability and little velocity. Forchheimer In (1992) Liao , 27 was In all of the discussed work, researchers consider heat and mass transmission features of Newtonian or non-Newtonian fluid at a time depended and a time independent extending surface, taking one or more physical characteristics. The main goal of this research is to investigate liquid film flow Williamson fluids over a stretched surface in the existence of magnetic field and thermal radiation. Keeping in view all these assumptions taken into the modelled problem and the similarity transformation method, the concerned PDEs are converted to non-linear ODEs, and the obtained, transformed equations are analytically solved using HAM.2x-direction. Also Consider the flow of non-Newtonian liquid film flow (considering Williamson fluid) with impact of thermal radiation over an unsteady porous stretching sheet. The coordinate system is chosen in such a way that the x-axis is parallel to the slit while the y-axis is perpendicular to the surface respectively . The x-aIn the interpretation of above expectations, the main leading equations are articulated as:Neglecting the higher order terms Eq. (5)Inserting By putting The accompanying boundary conditions here in Eqs. (2)Familiarizing the dimensionless 3For solution of the problem we implement the Homotopy Analysis Method to fin the solution of Eqs. The linear operators can be chosen asThe differential operators in The subjected boundary conditions for Eqs. Eq. (25)The resultant nonlinear operators areUsing the Taylor's series expansion to expand th order Eqs. th time we obtained the ith order deformation equations with respect to ith order deformation equationsDifferentiating ZeroThe resultant boundary conditions for Hence 4Here our interest is to analyze analytical solution of obtaining system of ordinary differential equations by Homotopy Analysis Method. When the series solution of the velocity and temperature profile are computed by HAM, the assisting parameters 5β throughout the flow motion. Increasing .The current research has been conceded out to study the flow of Williamson liquid film flow in a time dependent starching sheet with the impact of MHD and thermal radiation. The determination of this section is to examine the physical consequences of different embedding parameters on the velocity The numerical values of the surface temperature 6•Thermal boundary layer thickness reduces with rise of radiation parameter •The increasing values of •Increasing •For skin friction •It is notice that the strong magnetic field reduce the velocity he liquid films.•It is also concluded that liquid film flow is affected by the Lorentz's force.The conclusion of the present work is mainly focused on the behaviour of embedded parameters and solutions of the obtained results. The central concluded points are:Zahir Shah, Ebenezer Bonyah, Saeed Islam, Waris Khan, Mohammad Ishaq: Conceived and designed the analysis; Analyzed and interpreted the data; Contributed analysis tools or data; Wrote the paper.This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.The authors declare no conflict of interest.No additional information is available for this paper.