"Nobody does checkups back there": A qualitative study of refugees' healthcare needs in the United States from stakeholders' perspectives.

The number of refugees globally grew to 35.3 million in 2022, and many refugees are exposed to various health risks along their migration journey. As a result, they may arrive in host communities with numerous health issues, including communicable diseases and chronic and mental health conditions. Navigating the healthcare system in a host country proves to be a significant challenge for them, leading to delayed care. This qualitative study explored the convolute healthcare needs of refugees in the United States by soliciting insights from stakeholders involved in refugee resettlement and healthcare. In-depth interviews were conducted with fifteen stakeholders who work closely with refugees, including healthcare providers, cultural/clinical health navigators supporting refugees, staff from refugee resettlement agencies and governmental entities, and researchers studying refugee health. Following informed consent, interviews were audio-recorded, transcribed verbatim, and imported into MAXQDA 2022 (VERBI Software) for thematic analysis. The results revealed key themes, including the heterogeneity of refugee populations, limited awareness of preventive healthcare, high prevalence and suboptimal management of chronic conditions, complexity of the healthcare system, lack of follow-up, and language barriers. Further research is warranted concerning the long-term health of refugee populations in the United States. Additionally, more tailored programs involving peer educators are recommended to support refugee communities in navigating the complex healthcare system in the host country.

"And Now that I Feel Safe I'm Coming Out of Fight or Flight": A Qualitative Exploration of Challenges and Opportunities for Residents' Mental Health in Substance Use Recovery Housing.

Co-occurring mental health concerns are prevalent among substance use recovery housing residents. We sought to explore how residents with co-occurring mental health and substance use needs experience recovery housing. We conducted semi-structured interviews with residents (N = 92) in recovery homes across Texas and developed themes through thematic analysis. Residents note that living in a group home can exacerbate anxiety and paranoia, especially during periods of high turnover. Overwhelmingly, however, residents believe recovery housing improves their mental health. Residents use their shared lived experiences to support one another. Residents also express appreciation for the transition period offered by recovery housing, allowing them to solidify their recoveries before fully re-entering society. Participants describe recovery homes as a critical support for their co-occurring mental health and substance use concerns. These results provide key insights on how to better support mental health in recovery housing.

Leveraging qualitative approaches to guide sustainable international research collaborations.

Qualitative research approaches were used to launch an international research collaboration between the U. S. and Cambodia. Cambodian officials requested assistance in learning qualitative approaches to complement the research skills of Cambodian mental health providers. This article provides a description of how U. S. researchers responded to that request and engaged with Cambodian psychiatrists to explore mental health needs and interventions in both countries and initiate a sustainable relationship. The early focus on qualitative research methodologies may be an avenue that mitigates some of the challenges that can characterize international research. In this study, early communications involved developing a plan to teach qualitative methods while also collecting and analyzing data in both countries that would address the mental health concerns experienced by respective care providers. A case study exemplar was embedded with a scripted focus group guide to collect data from U. S. focus groups, then share with Cambodian psychiatrists. Components of hermeneutic phenomenological interviewing and descriptive content analysis were used to simultaneously teach and enact the research methods, gather data in both countries to analyze, and inspire participants to replicate the methods in their ongoing work. Cambodian psychiatrists were able to demonstrate competence in facilitating focus groups after being participant-observers. Researcher/practitioners from both U. S. and Cambodian teams gained new understandings about the mental health needs of their patients. The mutual engagement of a research focus is an effective way to establish cross-cultural relationships. The challenges of staying with stable teams over times remain, but the content shared and learned in a participatory structure yields understandings that cross cultural boundaries. Anticipated and unexpected challenges may be offset by an intention of reciprocity and mutual engagement. The use of qualitative methodologies, early and repeatedly, can facilitate relational understanding.

Amplitude Modulation Perception and Cortical Evoked Potentials in Children With Listening Difficulties and Their Typically Developing Peers.

PURPOSE: Amplitude modulations (AMs) are important for speech intelligibility, and deficits in speech intelligibility are a leading source of impairment in childhood listening difficulties (LiD). The present study aimed to explore the relationships between AM perception and speech-in-noise (SiN) comprehension in children and to determine whether deficits in AM processing contribute to childhood LiD. Evoked responses were used to parse the neural origins of AM processing. METHOD: Forty-one children with LiD and 44 typically developing children, ages 8-16 years, participated in the study. Behavioral AM depth thresholds were measured at 4 and 40 Hz. SiN tasks included the Listening in Spatialized Noise-Sentences Test (LiSN-S) and a coordinate response measure (CRM)-based task. Evoked responses were obtained during an AM change detection task using alternations between 4 and 40 Hz, including the N1 of the acoustic change complex, auditory steady-state response (ASSR), P300, and a late positive response (late potential [LP]). Maturational effects were explored via age correlations. RESULTS: Age correlated with 4-Hz AM thresholds, CRM separated talker scores, and N1 amplitude. Age-normed LiSN-S scores obtained without spatial or talker cues correlated with age-corrected 4-Hz AM thresholds and area under the LP curve. CRM separated talker scores correlated with AM thresholds and area under the LP curve. Most behavioral measures of AM perception correlated with the signal-to-noise ratio and phase coherence of the 40-Hz ASSR. AM change response time also correlated with area under the LP curve. Children with LiD exhibited deficits with respect to 4-Hz thresholds, AM change accuracy, and area under the LP curve. CONCLUSIONS: The observed relationships between AM perception and SiN performance extend the evidence that modulation perception is important for understanding SiN in childhood. In line with this finding, children with LiD demonstrated poorer performance on some measures of AM perception, but their evoked responses implicated a primarily cognitive deficit. SUPPLEMENTAL MATERIAL: https://doi.org/10.23641/asha.25009103.

"Being here is saving my life": Resident experiences of living in recovery residences for people taking medication for an opioid use disorder.

INTRODUCTION: Medications for opioid use disorder (MOUD) are an effective treatment for addressing opioid use disorder. Despite MOUD's demonstrated effectiveness, MOUD-related stigma is prevalent throughout many recovery communities and subsequently limits persons taking MOUD access to recovery supports, including recovery housing. While recovery residences that serve people taking MOUD could be a critical recovery support, they are limited in number and understudied. METHODS: We conducted in-depth interviews with 47 residents in medication-assisted recovery (MAR) living in 11 Texas-based recovery residences serving people taking MOUD to characterize residents' experiences and understand the impact that these homes had on their recovery. RESULTS: We found that many participants could not previously access recovery housing and other recovery supports due to MOUD-related stigma, thus recovery homes that supported people in MAR were considered a groundbreaking opportunity. Recovery residences provided participants with a space in which they did not feel judged for taking MOUD, which facilitated participants' connections with their fellow housemates. Subsequently, participants no longer had to hide their MAR pathway and could be transparent about taking MOUD among their recovery residence community. Last, recovery homes provided a supportive environment in which participants' internalized MOUD-related stigma could evolve into acceptance of their MAR pathway. CONCLUSIONS: Recovery residences that serve people in MAR provide a supportive, safe, nonjudgmental recovery environment in which residents develop relationships with other peers taking MOUD, share openly about their MAR, and are empowered to embrace their recovery pathway. These findings highlight the need for more recovery residences that are supportive of people taking MOUD as part of their recovery.

A maturational frequency discrimination deficit may explain developmental language disorder.

Auditory perceptual deficits are widely observed among children with developmental language disorder (DLD). Yet, the nature of these deficits and the extent to which they explain speech and language problems remain controversial. In this study, we hypothesize that disruption to the maturation of the basilar membrane may impede the optimization of the auditory pathway from brainstem to cortex, curtailing high-resolution frequency sensitivity and the efficient spectral decomposition and encoding of natural speech. A series of computational simulations involving deep convolutional neural networks that were trained to encode, recognize, and retrieve naturalistic speech are presented to demonstrate the strength of this account. These neural networks were built on top of biologically truthful inner ear models developed to model human cochlea function, which-in the key innovation of the present study-were scheduled to mature at different rates over time. Delaying cochlea maturation qualitatively replicated the linguistic behavior and neurophysiology of individuals with language learning difficulties in a number of ways, resulting in (a) delayed language acquisition profiles, (b) lower spoken word recognition accuracy, (c) word finding and retrieval difficulties, (d) "fuzzy" and intersecting speech encodings and signatures of immature neural optimization, and (e) emergent working memory and attentional deficits. These simulations illustrate many negative cascading effects that a primary maturational frequency discrimination deficit may have on early language development and generate precise and testable hypotheses for future research into the nature and cost of auditory processing deficits in children with language learning difficulties. (PsycInfo Database Record (c) 2024 APA, all rights reserved).

Amplitude modulation perception and cortical evoked potentials in children with listening difficulties and their typically-developing peers.

Purpose: Amplitude modulations (AM) are important for speech intelligibility, and deficits in speech intelligibility are a leading source of impairment in childhood listening difficulties (LiD). The present study aimed to explore the relationships between AM perception and speech-in-noise (SiN) comprehension in children and to determine whether deficits in AM processing contribute to childhood LiD. Evoked responses were used to parse the neural origin of AM processing. Method: Forty-one children with LiD and forty-four typically-developing children, ages 8-16 y.o., participated in the study. Behavioral AM depth thresholds were measured at 4 and 40 Hz. SiN tasks included the LiSN-S and a Coordinate Response Measure (CRM)-based task. Evoked responses were obtained during an AM Change detection task using alternations between 4 and 40 Hz, including the N1 of the acoustic change complex, auditory steady-state response (ASSR), P300, and a late positive response (LP). Maturational effects were explored via age correlations. Results: Age correlated with 4 Hz AM thresholds, CRM Separated Talker scores, and N1 amplitude. Age-normed LiSN-S scores obtained without spatial or talker cues correlated with age-corrected 4 Hz AM thresholds and area under the LP curve. CRM Separated Talker scores correlated with AM thresholds and area under the LP curve. Most behavioral measures of AM perception correlated with the SNR and phase coherence of the 40 Hz ASSR. AM Change RT also correlated with area under the LP curve. Children with LiD exhibited deficits with respect to 4 Hz thresholds, AM Change accuracy, and area under the LP curve. Conclusions: The observed relationships between AM perception and SiN performance extend the evidence that modulation perception is important for understanding SiN in childhood. In line with this finding, children with LiD demonstrated poorer performance on some measures of AM perception, but their evoked responses implicated a primarily cognitive deficit.

Targeting Cytotoxic Agents through EGFR-Mediated Covalent Binding and Release.

A major drawback of cytotoxic chemotherapy is the lack of selectivity toward noncancerous cells. The targeted delivery of cytotoxic drugs to tumor cells is a longstanding goal in cancer research. We proposed that covalent inhibitors could be adapted to deliver cytotoxic agents, conjugated to the ß-position of the Michael acceptor, via an addition-elimination mechanism promoted by covalent binding. Studies on model systems showed that conjugated 5-fluorouracil (5FU) could be released upon thiol addition in relevant time scales. A series of covalent epidermal growth factor receptor (EGFR) inhibitors were synthesized as their 5FU derivatives. Achieving the desired release of 5FU was demonstrated to depend on the electronics and geometry of the compounds. Mass spectrometry and NMR studies demonstrated an anilinoquinazoline acrylate ester conjugate bound to EGFR with the release of 5FU. This work establishes that acrylates can be used to release conjugated molecules upon covalent binding to proteins and could be used to develop targeted therapeutics.

Conformational study into N-alkyl-N'-aryl ureas to inform drug discovery.

Ureas are an important functional group in small molecule drugs as well as having wider applications in organic chemistry. Understanding of their conformation is of critical importance for rational design of urea-containing bioactive compounds. Whilst the conformational preferences of biaryl ureas have been extensively studied, very little attention has been paid to alkylated analogues. We carried out a systematic study of N-aryl (phenyl and pyridyl)-N'-cyclopentyl ureas with differing N-methylation patterns using Well Tempered Metadynamics at a semi-empirical level in implicit water (GBSA) using Well-Tempered Metadynamics to generate their conformational free-energy landscapes. Geometries and energetics of the most relevant configurations are further refined using DFT level of theory. Validation for the computation was obtained by synthesis of all 8 analogues followed by conformational studies by X-ray crystallography and NMR. These findings reveal that the methylation pattern significantly affects the conformational preference of the system. Most notably, N-phenyl-N'-cyclopentyl urea is shown to adopt both the trans-trans, and cis-trans conformations with equal energy and that the cis-trans conformation can be significantly stabilised by the presence of an internal hydrogen bond to the N'-hydrogen. This study will be of utility for the design of N-alkyl-N'-aryl ureas as drug candidates.

A Comparison of HPSP and USU Graduates' Preparation for Residency.

INTRODUCTION: Military medical students enter residency through two main pathways: (1) The Uniformed Services University (USU) and (2) the Armed Services Health Professions Scholarship Program (HPSP). The purpose of this study was to compare how these two pathways prepare military medical students for residency. METHODS: We conducted semi-structured interviews with 18 experienced military residency program directors (PDs) in order to explore their perceptions of the preparedness of USU and HPSP graduates. We used a transcendental phenomenological qualitative research design to bracket our biases and guide our data analysis. Our research team coded each of the interview transcripts. We then organized these codes into themes, which served as the results of our study. RESULTS: Five themes emerged from our data regarding the residents' preparedness: (1) Ability to navigate the military culture, (2) understanding of the military's medical mission, (3) clinical preparation, (4) ability to navigate the Military Health System (MHS), and (5) teamwork. The PDs described how USU graduates better understand the military's medical mission and are more easily able to navigate the military culture and the MHS because of their lived experiences during military medical school. They also discussed the various levels of clinical preparation of HPSP graduates, in contrast to the USU graduates' more consistent skills and abilities. Finally, the PDs believed both groups to be strong team players. CONCLUSIONS: USU students were consistently prepared for a strong start to residency because of their military medical school training. HPSP students often experienced a steep learning curve because of the newness of the military culture and MHS.

Speech cortical activation and connectivity in typically developing children and those with listening difficulties.

Listening difficulties (LiD) in people who have normal audiometry are a widespread but poorly understood form of hearing impairment. Recent research suggests that childhood LiD are cognitive rather than auditory in origin. We examined decoding of sentences using a novel combination of behavioral testing and fMRI with 43 typically developing children and 42 age matched (6-13 years old) children with LiD, categorized by caregiver report (ECLiPS). Both groups had clinically normal hearing. For sentence listening tasks, we found no group differences in fMRI brain cortical activation by increasingly complex speech stimuli that progressed in emphasis from phonology to intelligibility to semantics. Using resting state fMRI, we examined the temporal connectivity of cortical auditory and related speech perception networks. We found significant group differences only in cortical connections engaged when processing more complex speech stimuli. The strength of the affected connections was related to the children's performance on tests of dichotic listening, speech-in-noise, attention, memory and verbal vocabulary. Together, these results support the novel hypothesis that childhood LiD reflects difficulties in language rather than in auditory or phonological processing.

Build-Couple-Transform: A Paradigm for Lead-like Library Synthesis with Scaffold Diversity.

High-throughput screening provides one of the most common ways of finding hit compounds. Lead-like libraries, in particular, provide hits with compatible functional groups and vectors for structural elaboration and physical properties suitable for optimization. Library synthesis approaches can lead to a lack of chemical diversity because they employ parallel derivatization of common building blocks using single reaction types. We address this problem through a "build-couple-transform" paradigm for the generation of lead-like libraries with scaffold diversity. Nineteen transformations of a 4-oxo-2-butenamide scaffold template were optimized, including 1,4-cyclizations, 3,4-cyclizations, reductions, and 1,4-additions. A pool-transformation approach efficiently explored the scope of these transformations for nine different building blocks and synthesized a >170-member library with enhanced chemical space coverage and favorable drug-like properties. Screening revealed hits against CDK2. This work establishes the build-couple-transform concept for the synthesis of lead-like libraries and provides a differentiated approach to libraries with significantly enhanced scaffold diversity.

Adaptive Hearing Aid Benefit in Children With Mild/Moderate Hearing Loss: A Registered, Double-Blind, Randomized Clinical Trial.

OBJECTIVES: We completed a registered double-blind randomized control trial to compare acclimatization to two hearing aid fitting algorithms by experienced pediatric hearing aid users with mild to moderate hearing loss. We hypothesized that extended use (up to 13 months) of an adaptive algorithm with integrated directionality and noise reduction, OpenSound Navigator (OSN), would result in improved performance on auditory, cognitive, academic, and caregiver- or self-report measures compared with a control, omnidirectional algorithm (OMNI). DESIGN: Forty children aged 6 to 13 years with mild to moderate/severe symmetric sensorineural hearing loss completed this study. They were all experienced hearing aid users and were recruited through the Cincinnati Children's Hospital Medical Center Division of Audiology. The children were divided into 20 pairs based on similarity of age (within 1 year) and hearing loss (level and configuration). Individuals from each pair were randomly assigned to either an OSN (experimental) or OMNI (control) fitting algorithm group. Each child completed an audiology evaluation, hearing aid fitting using physically identical Oticon OPN hearing aids, follow-up audiological appointment, and 2 research visits up to 13 months apart. Research visit outcome measures covered speech perception (in quiet and in noise), novel grammar and word learning, cognition, academic ability, and caregiver report of listening behaviors. Analysis of outcome differences between visits, groups, ages, conditions and their interactions used linear mixed models. Between 22 and 39 children provided useable data for each task. RESULTS: Children using the experimental (OSN) algorithm did not show any significant performance differences on the outcome measures compared with those using the control (OMNI) algorithm. Overall performance of all children in the study increased across the duration of the trial on word repetition in noise, sentence repetition in quiet, and caregivers' assessment of hearing ability. There was a significant negative relationship between age at first hearing aid use, final Reading and Mathematical ability, and caregiver rated speech hearing. A significant positive relationship was found between daily hearing aid use and study-long change in performance on the Flanker test of inhibitory control and attention. Logged daily use of hearing aids related to caregiver rated spatial hearing. All results controlled for age at testing/evaluation and false discovery rate. CONCLUSIONS: Use of the experimental (OSN) algorithm neither enhanced nor reduced performance on auditory, cognitive, academic or caregiver report measures compared with the control (OMNI) algorithm. However, prolonged hearing aid use led to benefits in hearing, academic skills, attention, and caregiver evaluation.

Listening Difficulties in Children With Normal Audiograms: Relation to Hearing and Cognition.

OBJECTIVES: Children presenting at audiology services with caregiver-reported listening difficulties often have normal audiograms. The appropriate approach for the further assessment and clinical management of these children is currently unclear. In this Sensitive Indicators of Childhood Listening Difficulties (SICLiD) study, we assessed listening ability using a reliable and validated caregiver questionnaire (the Evaluation of Children's Listening and Processing Skills [ECLiPS]) in a large (n = 146) and heterogeneous sample of 6- to 13-year-old children with normal audiograms. Scores on the ECLiPS were related to a multifaceted laboratory assessment of the children's audiological, psycho- and physiological-acoustic, and cognitive abilities. This report is an overview of the SICLiD study and focuses on the children's behavioral performance. The overall goals of SICLiD were to understand the auditory and other neural mechanisms underlying childhood listening difficulties and translate that understanding into clinical assessment and, ultimately, intervention. DESIGN: Cross-sectional behavioral assessment of children with "listening difficulties" and an age-matched "typically developing" control group. Caregivers completed the ECLiPS, and the resulting total standardized composite score formed the basis of further descriptive statistics, univariate, and multivariate modeling of experimental data. RESULTS: All scores of the ECLiPS, the SCAN-3:C, a standardized clinical test suite for auditory processing, and the National Institutes of Health (NIH) Cognition Toolbox were significantly lower for children with listening difficulties than for their typically developing peers using group comparisons via t-tests and Wilcoxon Rank-Sum tests. A similar effect was observed on the Listening in Spatialized Noise-Sentences (LiSN-S) test for speech sentence-in-noise intelligibility but only reached significance for the Low Cue and High Cue conditions and the Talker Advantage derived score. Stepwise regression to examine the factors contributing to the ECLiPS Total scaled score (pooled across groups) yielded a model that explained 42% of its variance based on the SCAN-3:C composite, LiSN-S Talker Advantage, and the NIH Toolbox Picture Vocabulary, and Dimensional Change Card Sorting scores (F[4, 95] = 17.35, p < 0.001). High correlations were observed between many test scores including the ECLiPS, SCAN-3:C, and NIH Toolbox composite measures. LiSN-S Advantage measures generally correlated weakly and nonsignificantly with non-LiSN-S measures. However, a significant interaction was found between extended high-frequency threshold and LiSN-S Talker Advantage. CONCLUSIONS: Children with listening difficulties but normal audiograms have problems with the cognitive processing of auditory and nonauditory stimuli that include both fluid and crystallized reasoning. Analysis of poor performance on the LiSN-S Talker Advantage measure identified subclinical hearing loss as a minor contributing factor to talker segregation. Beyond auditory tests, evaluations of children with complaints of listening difficulties should include standardized caregiver observations and consideration of broad cognitive abilities.

An Alkynylpyrimidine-Based Covalent Inhibitor That Targets a Unique Cysteine in NF-κB-Inducing Kinase.

NF-κB-inducing kinase (NIK) is a key enzyme in the noncanonical NF-κB pathway, of interest in the treatment of a variety of diseases including cancer. Validation of NIK as a drug target requires potent and selective inhibitors. The protein contains a cysteine residue at position 444 in the back pocket of the active site, unique within the kinome. Analysis of existing inhibitor scaffolds and early structure-activity relationships (SARs) led to the design of C444-targeting covalent inhibitors based on alkynyl heterocycle warheads. Mass spectrometry provided proof of the covalent mechanism, and the SAR was rationalized by computational modeling. Profiling of more potent analogues in tumor cell lines with constitutively activated NIK signaling induced a weak antiproliferative effect, suggesting that kinase inhibition may have limited impact on cancer cell growth. This study shows that alkynyl heterocycles are potential cysteine traps, which may be employed where common Michael acceptors, such as acrylamides, are not tolerated.

Short-Term Pediatric Acclimatization to Adaptive Hearing Aid Technology.

Purpose This exploratory study assessed the perceptual, cognitive, and academic learning effects of an adaptive, integrated, directionality, and noise reduction hearing aid program in pediatric users. Method Fifteen pediatric hearing aid users (6-12 years old) received new bilateral, individually fitted Oticon Opn hearing aids programmed with OpenSound Navigator (OSN) processing. Word recognition in noise, sentence repetition in quiet, nonword repetition, vocabulary learning, selective attention, executive function, memory, and reading and mathematical abilities were measured within 1 week of the initial hearing aid fitting and 2 months post fit. Caregivers completed questionnaires assessing their child's listening and communication abilities prior to study enrollment and after 2 months of using the study hearing aids. Results Caregiver reporting indicated significant improvements in speech and sound perception, spatial sound awareness, and the ability to participate in conversations. However, there was no positive change in performance in any of the measured skills. Mathematical scores significantly declined after 2 months. Conclusions OSN provided a perceived improvement in functional benefit, compared to their previous hearing aids, as reported by caregivers. However, there was no positive change in listening skills, cognition, and academic success after 2 months of using OSN. Findings may have been impacted by reporter bias, limited sample size, and a relatively short trial period. This study took place during the summer when participants were out of school, which may have influenced the decline in mathematical scores. The results support further exploration with age- and audiogram-matched controls, larger sample sizes, and longer test-retest intervals that correspond to the academic school year.

Peripheral Auditory Involvement in Childhood Listening Difficulty.

OBJECTIVES: This study tested the hypothesis that undetected peripheral hearing impairment occurs in children with idiopathic listening difficulties (LiDs), as reported by caregivers using the Evaluation of Children"s Listening and Processing Skills (ECLiPS) validated questionnaire, compared with children with typically developed (TD) listening abilities. DESIGN: Children with LiD aged 6-14 years old (n = 60, mean age = 9.9 yr) and 54 typical age matched children were recruited from audiology clinical records and from IRB-approved advertisements at hospital locations and in the local and regional areas. Both groups completed standard and extended high-frequency (EHF) pure-tone audiometry, wideband absorbance tympanometry and middle ear muscle reflexes, distortion product and chirp transient evoked otoacoustic emissions. Univariate and multivariate mixed models and multiple regression analysis were used to examine group differences and continuous performance, as well as the influence of demographic factors and pressure equalization (PE) tube history. RESULTS: There were no significant group differences between the LiD and TD groups for any of the auditory measures tested. However, analyses across all children showed that EHF hearing thresholds, wideband tympanometry, contralateral middle ear muscle reflexes, distortion product, and transient-evoked otoacoustic emissions were related to a history of PE tube surgery. The physiologic measures were also associated with EHF hearing loss, secondary to PE tube history. CONCLUSIONS: Overall, the results of this study in a sample of children with validated LiD compared with a TD group matched for age and sex showed no significant differences in peripheral function using highly sensitive auditory measures. Histories of PE tube surgery were significantly related to EHF hearing and to a range of physiologic measures in the combined sample.