Kaufman-Mckusick Syndrome

Translated BLAST runs were carried out on the wheat CDS sequences in the Triticeae full-length CDS database using the Brachypodium MPK and MKK sequences identified by Chen et al. [11 (link)]. Similar searches were carried out using the TSA databases (in-house triticale database and the rye NCBI BioProject PRJDB2278). Collected sequences were accepted based on their alignment with Brachypodium orthologues or if they carried MPK or MKK consensus sequences including the conserved residues D(L/I/V)K in the active site motif, and the S/TxxxxxS/T activation loop for MKKs and TxY for MPKs. Nucleotide sequences were then used to search the Ensembl genomic database for wheat, barley, A. tauschii and T. urartu homologues. Domain analysis was carried out with ExPASy PROSITE (http://prosite.expasy.org/), which identified the protein kinase domain (PS50011) and related signature sequences: MAP kinase signature (PS01351); PK ATP binding signature (PS00107); S/T PK active site signature (PS00108). Additional conserved domains specific to plant MPKs as defined by Mohanta et al. [18 (link)] were also assessed by visual sequence analysis.
Multiple Expectation maximization for Motif Elicitation (MEME) analysis was carried out with the intent of generating sequence distribution figures, rather than identifying domains. Conserved domains of interest from alignments were used as input sequences in the MEME freeware (http://meme-suite.org/tools/meme).

A near isogenic line WL711+Lr57 (T756; pau16062) containing leaf rust resistance gene Lr57 introgressed from wild wheat species A. geniculata in wheat cv. WL711 background and the recipient parent WL711 were used for studying the differential expression (DE) under compatible and incompatible interactions with P. triticina pathotype 77-5. The near isogenic line was developed by crossing disomic substitution line DS5M (5D) in WL711 background with Chinese Spring stock having suppressor for Ph1 locus for induction of homoeologous pairing. The F₁ was again crossed and backcrossed with the recurrent parent WL711. Leaf and stripe rust resistant BC₃F₁ plants were selfed to generate BC₃F₁₅ introgression line which was used for transcriptome sequencing. The development of introgression lines carrying Lr57 has also been described in Kuraparthy et al. (2007 (link)).
WL711 and WL711+Lr57 were planted in sand:cocopeat mix in plastic trays in three replicates each and grown under 16:8 h light and dark cycle. First leaf of the 7-day-old seedlings of WL711 and WL711+Lr57 were inoculated with P. triticina race 77-5 under screen house conditions in three biological replicates. Leaf samples were collected for total RNA extraction at five different time intervals (0, 12, 24, 48, and 72 h post inoculation; HPI) and stored in RNAlater® solution (Ambion) for further processing. One set of inoculated seedlings of WL711 and WL711+Lr57 were grown for another fortnight to record disease development. Five time points for sampling were chosen based on the life cycle stages of the leaf rust pathogen (Hu and Rijkenberg, 1998 (link)). Initial time point selection of 0 HPI represent the contol mock inoculated leaves, while 12 and 24 HPI stages symbolized the development of haustorial mother cell and haustoria between mesophyll cells, respectively. The later two time scales (48 and 72 HPI) correspond to the further development in secondary hyphae, HMCs and huastoria leading to the development of networked fungal mycelia.

The initial sampling method entailed defining sampling criteria for potential participants. Criterion sampling was used purposefully to select participants who met the prespecified eligibility criteria [29 , 71 ]. There were two groups of participants with the first being healthcare professionals (HCPs) who provided direct care either at the hospital and community level and healthcare managers (HCMs) in managerial and administrative positions at the hospital, district, and regional levels. HCPs who were still undergoing basic training or had been in their current position for less than one month were not considered eligible. The second group included mothers and relatives whose LBW infants were admitted for at least 24 h to the NICU and later discharged home not longer than eight weeks prior to the interview. The time frame of 24 h was chosen because in LMICs, healthy newborns, including LBW infants are often discharged early due to overcrowded neonatal units, mothers’ having older children at home to care for, lack of support and financial constraints faced by parents [4 (link), 24 (link), 35 (link)]. The mothers and relatives of LBW infants who had been transferred to a higher-level care facility after being admitted at the NICU and had not returned to the NICU were not recruited.
Relatives other than mothers and fathers are often substantially involved in the care of LBW infants in Ghana [3 (link), 77 (link)]. Therefore, other caregivers pointed out by the mothers to be significantly involved in caregiving for their LBW infants were also recruited.
The caregivers in this study were purposively sampled without regard to their educational status. All participants were required to speak the local language (Ewe) or English.
After the initial contact was established through a gatekeeper, interviews were first conducted with the HCPs at the NICU. Further interviews were held with facility-based HCPs directly involved in clinical care and HCMs responsible for managerial tasks concerning LBW infants. Later, HCPs from four CHPS zones at the primary care level and HCMs from the district and regional levels were interviewed. All interviews with HCPs and HCMs were conducted in English. Families of LBW infants were called and informed about the study. After agreeing to participate, those unable to speak English were interviewed in the local language. All prospective participants who were contacted consented with none unwilling to participate nor withdrawing the consent along the line. After initial sampling theoretical sampling followed. Theoretical sampling is an open selection of data that helps to decide what subsequent data to collect to develop a theory [28 (link), 37 ]. Subsequently, additional HCPs and HMCs at different levels of care were recruited after earlier interviews revealed that they played a major role in the care continuum. Sampling continued as theoretical categories were developing. This involved selecting new participants or re-interviewing earlier participants. For example, to expand the category concerning the discharge process, two additional health professionals at the hospital and another two at community level were interviewed. The follow up interviews helped to specify the properties of selected categories and to get more in-depth information where details were still lacking. Theoretical saturation was reached, when new data collected did not reveal any new properties to the theoretical categories nor provided any further insights about the emerging theory [25 ].

A CCK-8 assay was used to analyze the inhibitory effect of DHT on the viability of HMCs. HMCs (5× 10³ cells/well) were inoculated into 96-well plates. After incubation, the cell culture medium was substituted with fresh medium containing various concentrations (0.1, 1, 5, 10, 100 µM) of DHT and incubated for 48 h. Next, 10 μL of CCK-8 solution was added to every well, after which the cells were incubated at 37°C for 1 h. Each well’s optical density (OD) was calculated at 450 nm using a spectrophotometer 1510 (Thermo Fisher Scientific, USA).